anno_start	anno_end	anno_text	entity_type	sentence	section
12	15	DNA	chemical	Reversal of DNA damage induced Topoisomerase 2 DNA–protein crosslinks by Tdp2	TITLE
31	46	Topoisomerase 2	protein_type	Reversal of DNA damage induced Topoisomerase 2 DNA–protein crosslinks by Tdp2	TITLE
47	50	DNA	chemical	Reversal of DNA damage induced Topoisomerase 2 DNA–protein crosslinks by Tdp2	TITLE
73	77	Tdp2	protein	Reversal of DNA damage induced Topoisomerase 2 DNA–protein crosslinks by Tdp2	TITLE
0	9	Mammalian	taxonomy_domain	Mammalian Tyrosyl-DNA phosphodiesterase 2 (Tdp2) reverses Topoisomerase 2 (Top2) DNA–protein crosslinks triggered by Top2 engagement of DNA damage or poisoning by anticancer drugs.	ABSTRACT
10	41	Tyrosyl-DNA phosphodiesterase 2	protein	Mammalian Tyrosyl-DNA phosphodiesterase 2 (Tdp2) reverses Topoisomerase 2 (Top2) DNA–protein crosslinks triggered by Top2 engagement of DNA damage or poisoning by anticancer drugs.	ABSTRACT
43	47	Tdp2	protein	Mammalian Tyrosyl-DNA phosphodiesterase 2 (Tdp2) reverses Topoisomerase 2 (Top2) DNA–protein crosslinks triggered by Top2 engagement of DNA damage or poisoning by anticancer drugs.	ABSTRACT
58	73	Topoisomerase 2	protein_type	Mammalian Tyrosyl-DNA phosphodiesterase 2 (Tdp2) reverses Topoisomerase 2 (Top2) DNA–protein crosslinks triggered by Top2 engagement of DNA damage or poisoning by anticancer drugs.	ABSTRACT
75	79	Top2	protein_type	Mammalian Tyrosyl-DNA phosphodiesterase 2 (Tdp2) reverses Topoisomerase 2 (Top2) DNA–protein crosslinks triggered by Top2 engagement of DNA damage or poisoning by anticancer drugs.	ABSTRACT
81	84	DNA	chemical	Mammalian Tyrosyl-DNA phosphodiesterase 2 (Tdp2) reverses Topoisomerase 2 (Top2) DNA–protein crosslinks triggered by Top2 engagement of DNA damage or poisoning by anticancer drugs.	ABSTRACT
117	121	Top2	protein_type	Mammalian Tyrosyl-DNA phosphodiesterase 2 (Tdp2) reverses Topoisomerase 2 (Top2) DNA–protein crosslinks triggered by Top2 engagement of DNA damage or poisoning by anticancer drugs.	ABSTRACT
136	139	DNA	chemical	Mammalian Tyrosyl-DNA phosphodiesterase 2 (Tdp2) reverses Topoisomerase 2 (Top2) DNA–protein crosslinks triggered by Top2 engagement of DNA damage or poisoning by anticancer drugs.	ABSTRACT
0	4	Tdp2	protein	Tdp2 deficiencies are linked to neurological disease and cellular sensitivity to Top2 poisons.	ABSTRACT
81	85	Top2	protein_type	Tdp2 deficiencies are linked to neurological disease and cellular sensitivity to Top2 poisons.	ABSTRACT
18	42	X-ray crystal structures	evidence	Herein, we report X-ray crystal structures of ligand-free Tdp2 and Tdp2-DNA complexes with alkylated and abasic DNA that unveil a dynamic Tdp2 active site lid and deep substrate binding trench well-suited for engaging the diverse DNA damage triggers of abortive Top2 reactions.	ABSTRACT
46	57	ligand-free	protein_state	Herein, we report X-ray crystal structures of ligand-free Tdp2 and Tdp2-DNA complexes with alkylated and abasic DNA that unveil a dynamic Tdp2 active site lid and deep substrate binding trench well-suited for engaging the diverse DNA damage triggers of abortive Top2 reactions.	ABSTRACT
58	62	Tdp2	protein	Herein, we report X-ray crystal structures of ligand-free Tdp2 and Tdp2-DNA complexes with alkylated and abasic DNA that unveil a dynamic Tdp2 active site lid and deep substrate binding trench well-suited for engaging the diverse DNA damage triggers of abortive Top2 reactions.	ABSTRACT
67	75	Tdp2-DNA	complex_assembly	Herein, we report X-ray crystal structures of ligand-free Tdp2 and Tdp2-DNA complexes with alkylated and abasic DNA that unveil a dynamic Tdp2 active site lid and deep substrate binding trench well-suited for engaging the diverse DNA damage triggers of abortive Top2 reactions.	ABSTRACT
112	115	DNA	chemical	Herein, we report X-ray crystal structures of ligand-free Tdp2 and Tdp2-DNA complexes with alkylated and abasic DNA that unveil a dynamic Tdp2 active site lid and deep substrate binding trench well-suited for engaging the diverse DNA damage triggers of abortive Top2 reactions.	ABSTRACT
130	137	dynamic	protein_state	Herein, we report X-ray crystal structures of ligand-free Tdp2 and Tdp2-DNA complexes with alkylated and abasic DNA that unveil a dynamic Tdp2 active site lid and deep substrate binding trench well-suited for engaging the diverse DNA damage triggers of abortive Top2 reactions.	ABSTRACT
138	142	Tdp2	protein	Herein, we report X-ray crystal structures of ligand-free Tdp2 and Tdp2-DNA complexes with alkylated and abasic DNA that unveil a dynamic Tdp2 active site lid and deep substrate binding trench well-suited for engaging the diverse DNA damage triggers of abortive Top2 reactions.	ABSTRACT
143	158	active site lid	structure_element	Herein, we report X-ray crystal structures of ligand-free Tdp2 and Tdp2-DNA complexes with alkylated and abasic DNA that unveil a dynamic Tdp2 active site lid and deep substrate binding trench well-suited for engaging the diverse DNA damage triggers of abortive Top2 reactions.	ABSTRACT
168	192	substrate binding trench	site	Herein, we report X-ray crystal structures of ligand-free Tdp2 and Tdp2-DNA complexes with alkylated and abasic DNA that unveil a dynamic Tdp2 active site lid and deep substrate binding trench well-suited for engaging the diverse DNA damage triggers of abortive Top2 reactions.	ABSTRACT
230	233	DNA	chemical	Herein, we report X-ray crystal structures of ligand-free Tdp2 and Tdp2-DNA complexes with alkylated and abasic DNA that unveil a dynamic Tdp2 active site lid and deep substrate binding trench well-suited for engaging the diverse DNA damage triggers of abortive Top2 reactions.	ABSTRACT
262	266	Top2	protein_type	Herein, we report X-ray crystal structures of ligand-free Tdp2 and Tdp2-DNA complexes with alkylated and abasic DNA that unveil a dynamic Tdp2 active site lid and deep substrate binding trench well-suited for engaging the diverse DNA damage triggers of abortive Top2 reactions.	ABSTRACT
23	27	Tdp2	protein	Modeling of a proposed Tdp2 reaction coordinate, combined with mutagenesis and biochemical studies support a single Mg2+-ion mechanism assisted by a phosphotyrosyl-arginine cation-π interface.	ABSTRACT
63	74	mutagenesis	experimental_method	Modeling of a proposed Tdp2 reaction coordinate, combined with mutagenesis and biochemical studies support a single Mg2+-ion mechanism assisted by a phosphotyrosyl-arginine cation-π interface.	ABSTRACT
79	98	biochemical studies	experimental_method	Modeling of a proposed Tdp2 reaction coordinate, combined with mutagenesis and biochemical studies support a single Mg2+-ion mechanism assisted by a phosphotyrosyl-arginine cation-π interface.	ABSTRACT
116	120	Mg2+	chemical	Modeling of a proposed Tdp2 reaction coordinate, combined with mutagenesis and biochemical studies support a single Mg2+-ion mechanism assisted by a phosphotyrosyl-arginine cation-π interface.	ABSTRACT
149	191	phosphotyrosyl-arginine cation-π interface	site	Modeling of a proposed Tdp2 reaction coordinate, combined with mutagenesis and biochemical studies support a single Mg2+-ion mechanism assisted by a phosphotyrosyl-arginine cation-π interface.	ABSTRACT
22	26	Tdp2	protein	We further identify a Tdp2 active site SNP that ablates Tdp2 Mg2+ binding and catalytic activity, impairs Tdp2 mediated NHEJ of tyrosine blocked termini, and renders cells sensitive to the anticancer agent etoposide.	ABSTRACT
27	38	active site	site	We further identify a Tdp2 active site SNP that ablates Tdp2 Mg2+ binding and catalytic activity, impairs Tdp2 mediated NHEJ of tyrosine blocked termini, and renders cells sensitive to the anticancer agent etoposide.	ABSTRACT
48	55	ablates	protein_state	We further identify a Tdp2 active site SNP that ablates Tdp2 Mg2+ binding and catalytic activity, impairs Tdp2 mediated NHEJ of tyrosine blocked termini, and renders cells sensitive to the anticancer agent etoposide.	ABSTRACT
56	60	Tdp2	protein	We further identify a Tdp2 active site SNP that ablates Tdp2 Mg2+ binding and catalytic activity, impairs Tdp2 mediated NHEJ of tyrosine blocked termini, and renders cells sensitive to the anticancer agent etoposide.	ABSTRACT
61	65	Mg2+	chemical	We further identify a Tdp2 active site SNP that ablates Tdp2 Mg2+ binding and catalytic activity, impairs Tdp2 mediated NHEJ of tyrosine blocked termini, and renders cells sensitive to the anticancer agent etoposide.	ABSTRACT
106	110	Tdp2	protein	We further identify a Tdp2 active site SNP that ablates Tdp2 Mg2+ binding and catalytic activity, impairs Tdp2 mediated NHEJ of tyrosine blocked termini, and renders cells sensitive to the anticancer agent etoposide.	ABSTRACT
128	136	tyrosine	residue_name	We further identify a Tdp2 active site SNP that ablates Tdp2 Mg2+ binding and catalytic activity, impairs Tdp2 mediated NHEJ of tyrosine blocked termini, and renders cells sensitive to the anticancer agent etoposide.	ABSTRACT
206	215	etoposide	chemical	We further identify a Tdp2 active site SNP that ablates Tdp2 Mg2+ binding and catalytic activity, impairs Tdp2 mediated NHEJ of tyrosine blocked termini, and renders cells sensitive to the anticancer agent etoposide.	ABSTRACT
61	65	Tdp2	protein	Collectively, our results provide a structural mechanism for Tdp2 engagement of heterogeneous DNA damage that causes Top2 poisoning, and indicate that evaluation of Tdp2 status may be an important personalized medicine biomarker informing on individual sensitivities to chemotherapeutic Top2 poisons.	ABSTRACT
94	97	DNA	chemical	Collectively, our results provide a structural mechanism for Tdp2 engagement of heterogeneous DNA damage that causes Top2 poisoning, and indicate that evaluation of Tdp2 status may be an important personalized medicine biomarker informing on individual sensitivities to chemotherapeutic Top2 poisons.	ABSTRACT
117	121	Top2	protein_type	Collectively, our results provide a structural mechanism for Tdp2 engagement of heterogeneous DNA damage that causes Top2 poisoning, and indicate that evaluation of Tdp2 status may be an important personalized medicine biomarker informing on individual sensitivities to chemotherapeutic Top2 poisons.	ABSTRACT
165	169	Tdp2	protein	Collectively, our results provide a structural mechanism for Tdp2 engagement of heterogeneous DNA damage that causes Top2 poisoning, and indicate that evaluation of Tdp2 status may be an important personalized medicine biomarker informing on individual sensitivities to chemotherapeutic Top2 poisons.	ABSTRACT
287	291	Top2	protein_type	Collectively, our results provide a structural mechanism for Tdp2 engagement of heterogeneous DNA damage that causes Top2 poisoning, and indicate that evaluation of Tdp2 status may be an important personalized medicine biomarker informing on individual sensitivities to chemotherapeutic Top2 poisons.	ABSTRACT
8	11	DNA	chemical	Nuclear DNA compaction and the action of DNA and RNA polymerases create positive and negative DNA supercoiling—over- and under-winding of DNA strands, respectively—and the linking together (catenation) of DNA strands.	INTRO
41	44	DNA	chemical	Nuclear DNA compaction and the action of DNA and RNA polymerases create positive and negative DNA supercoiling—over- and under-winding of DNA strands, respectively—and the linking together (catenation) of DNA strands.	INTRO
49	64	RNA polymerases	protein_type	Nuclear DNA compaction and the action of DNA and RNA polymerases create positive and negative DNA supercoiling—over- and under-winding of DNA strands, respectively—and the linking together (catenation) of DNA strands.	INTRO
94	97	DNA	chemical	Nuclear DNA compaction and the action of DNA and RNA polymerases create positive and negative DNA supercoiling—over- and under-winding of DNA strands, respectively—and the linking together (catenation) of DNA strands.	INTRO
138	141	DNA	chemical	Nuclear DNA compaction and the action of DNA and RNA polymerases create positive and negative DNA supercoiling—over- and under-winding of DNA strands, respectively—and the linking together (catenation) of DNA strands.	INTRO
205	208	DNA	chemical	Nuclear DNA compaction and the action of DNA and RNA polymerases create positive and negative DNA supercoiling—over- and under-winding of DNA strands, respectively—and the linking together (catenation) of DNA strands.	INTRO
0	14	Topoisomerases	protein_type	Topoisomerases relieve topological DNA strain and entanglement to facilitate critical nuclear DNA transactions including DNA replication, transcription and cell division.	INTRO
35	38	DNA	chemical	Topoisomerases relieve topological DNA strain and entanglement to facilitate critical nuclear DNA transactions including DNA replication, transcription and cell division.	INTRO
94	97	DNA	chemical	Topoisomerases relieve topological DNA strain and entanglement to facilitate critical nuclear DNA transactions including DNA replication, transcription and cell division.	INTRO
121	124	DNA	chemical	Topoisomerases relieve topological DNA strain and entanglement to facilitate critical nuclear DNA transactions including DNA replication, transcription and cell division.	INTRO
4	13	mammalian	taxonomy_domain	The mammalian type II topoisomerases Top2α and Top2β enzymes generate transient, reversible DNA double strand breaks (DSBs) to drive topological transactions.	INTRO
14	36	type II topoisomerases	protein_type	The mammalian type II topoisomerases Top2α and Top2β enzymes generate transient, reversible DNA double strand breaks (DSBs) to drive topological transactions.	INTRO
37	42	Top2α	protein	The mammalian type II topoisomerases Top2α and Top2β enzymes generate transient, reversible DNA double strand breaks (DSBs) to drive topological transactions.	INTRO
47	52	Top2β	protein	The mammalian type II topoisomerases Top2α and Top2β enzymes generate transient, reversible DNA double strand breaks (DSBs) to drive topological transactions.	INTRO
92	95	DNA	chemical	The mammalian type II topoisomerases Top2α and Top2β enzymes generate transient, reversible DNA double strand breaks (DSBs) to drive topological transactions.	INTRO
17	21	Top2	protein_type	Reversibility of Top2 DNA cleavage reactions is facilitated by formation of covalent enzyme phosphotyrosyl linkages between the 5′-phosphate ends of the incised duplex and an active site Top2 tyrosine, resulting in Top2 cleavage complexes (Top2cc).	INTRO
22	25	DNA	chemical	Reversibility of Top2 DNA cleavage reactions is facilitated by formation of covalent enzyme phosphotyrosyl linkages between the 5′-phosphate ends of the incised duplex and an active site Top2 tyrosine, resulting in Top2 cleavage complexes (Top2cc).	INTRO
92	115	phosphotyrosyl linkages	ptm	Reversibility of Top2 DNA cleavage reactions is facilitated by formation of covalent enzyme phosphotyrosyl linkages between the 5′-phosphate ends of the incised duplex and an active site Top2 tyrosine, resulting in Top2 cleavage complexes (Top2cc).	INTRO
128	140	5′-phosphate	chemical	Reversibility of Top2 DNA cleavage reactions is facilitated by formation of covalent enzyme phosphotyrosyl linkages between the 5′-phosphate ends of the incised duplex and an active site Top2 tyrosine, resulting in Top2 cleavage complexes (Top2cc).	INTRO
175	186	active site	site	Reversibility of Top2 DNA cleavage reactions is facilitated by formation of covalent enzyme phosphotyrosyl linkages between the 5′-phosphate ends of the incised duplex and an active site Top2 tyrosine, resulting in Top2 cleavage complexes (Top2cc).	INTRO
187	191	Top2	protein_type	Reversibility of Top2 DNA cleavage reactions is facilitated by formation of covalent enzyme phosphotyrosyl linkages between the 5′-phosphate ends of the incised duplex and an active site Top2 tyrosine, resulting in Top2 cleavage complexes (Top2cc).	INTRO
192	200	tyrosine	residue_name	Reversibility of Top2 DNA cleavage reactions is facilitated by formation of covalent enzyme phosphotyrosyl linkages between the 5′-phosphate ends of the incised duplex and an active site Top2 tyrosine, resulting in Top2 cleavage complexes (Top2cc).	INTRO
215	219	Top2	protein_type	Reversibility of Top2 DNA cleavage reactions is facilitated by formation of covalent enzyme phosphotyrosyl linkages between the 5′-phosphate ends of the incised duplex and an active site Top2 tyrosine, resulting in Top2 cleavage complexes (Top2cc).	INTRO
240	246	Top2cc	complex_assembly	Reversibility of Top2 DNA cleavage reactions is facilitated by formation of covalent enzyme phosphotyrosyl linkages between the 5′-phosphate ends of the incised duplex and an active site Top2 tyrosine, resulting in Top2 cleavage complexes (Top2cc).	INTRO
4	10	Top2cc	complex_assembly	The Top2cc protein–DNA adduct is a unique threat to genomic integrity which must be resolved to prevent catastrophic Top2cc collisions with the cellular replication and transcription machineries.	INTRO
19	22	DNA	chemical	The Top2cc protein–DNA adduct is a unique threat to genomic integrity which must be resolved to prevent catastrophic Top2cc collisions with the cellular replication and transcription machineries.	INTRO
117	123	Top2cc	complex_assembly	The Top2cc protein–DNA adduct is a unique threat to genomic integrity which must be resolved to prevent catastrophic Top2cc collisions with the cellular replication and transcription machineries.	INTRO
30	34	Top2	protein_type	To promote cancer cell death, Top2 reactions are ‘poisoned’ by keystone pharmacological anticancer agents like etoposide, teniposide and doxorubicin.	INTRO
111	120	etoposide	chemical	To promote cancer cell death, Top2 reactions are ‘poisoned’ by keystone pharmacological anticancer agents like etoposide, teniposide and doxorubicin.	INTRO
122	132	teniposide	chemical	To promote cancer cell death, Top2 reactions are ‘poisoned’ by keystone pharmacological anticancer agents like etoposide, teniposide and doxorubicin.	INTRO
137	148	doxorubicin	chemical	To promote cancer cell death, Top2 reactions are ‘poisoned’ by keystone pharmacological anticancer agents like etoposide, teniposide and doxorubicin.	INTRO
13	17	Top2	protein_type	Importantly, Top2 is also poisoned when it engages abundant endogenous DNA damage not limited to but including ribonucleotides, abasic sites and alkylation damage such as exocyclic DNA adducts arising from bioactivation of the vinyl chloride carcinogen (Figure 1A).	INTRO
71	74	DNA	chemical	Importantly, Top2 is also poisoned when it engages abundant endogenous DNA damage not limited to but including ribonucleotides, abasic sites and alkylation damage such as exocyclic DNA adducts arising from bioactivation of the vinyl chloride carcinogen (Figure 1A).	INTRO
181	184	DNA	chemical	Importantly, Top2 is also poisoned when it engages abundant endogenous DNA damage not limited to but including ribonucleotides, abasic sites and alkylation damage such as exocyclic DNA adducts arising from bioactivation of the vinyl chloride carcinogen (Figure 1A).	INTRO
15	18	DNA	chemical	In the case of DNA damage-triggered Top2cc, compound DNA lesions arise that consist of the instigating lesion, and a DNA DSB bearing a bulky terminal 5′-linked Top2 DNA–protein crosslink.	INTRO
36	42	Top2cc	complex_assembly	In the case of DNA damage-triggered Top2cc, compound DNA lesions arise that consist of the instigating lesion, and a DNA DSB bearing a bulky terminal 5′-linked Top2 DNA–protein crosslink.	INTRO
53	56	DNA	chemical	In the case of DNA damage-triggered Top2cc, compound DNA lesions arise that consist of the instigating lesion, and a DNA DSB bearing a bulky terminal 5′-linked Top2 DNA–protein crosslink.	INTRO
117	120	DNA	chemical	In the case of DNA damage-triggered Top2cc, compound DNA lesions arise that consist of the instigating lesion, and a DNA DSB bearing a bulky terminal 5′-linked Top2 DNA–protein crosslink.	INTRO
160	164	Top2	protein_type	In the case of DNA damage-triggered Top2cc, compound DNA lesions arise that consist of the instigating lesion, and a DNA DSB bearing a bulky terminal 5′-linked Top2 DNA–protein crosslink.	INTRO
165	168	DNA	chemical	In the case of DNA damage-triggered Top2cc, compound DNA lesions arise that consist of the instigating lesion, and a DNA DSB bearing a bulky terminal 5′-linked Top2 DNA–protein crosslink.	INTRO
27	30	DNA	chemical	The chemical complexity of DNA damage-derived Top2cc necessitates that DNA repair machinery dedicated to resolving these lesions recognizes both DNA and protein, whilst accommodating diverse chemical structures that trap Top2cc.	INTRO
46	52	Top2cc	complex_assembly	The chemical complexity of DNA damage-derived Top2cc necessitates that DNA repair machinery dedicated to resolving these lesions recognizes both DNA and protein, whilst accommodating diverse chemical structures that trap Top2cc.	INTRO
71	74	DNA	chemical	The chemical complexity of DNA damage-derived Top2cc necessitates that DNA repair machinery dedicated to resolving these lesions recognizes both DNA and protein, whilst accommodating diverse chemical structures that trap Top2cc.	INTRO
145	148	DNA	chemical	The chemical complexity of DNA damage-derived Top2cc necessitates that DNA repair machinery dedicated to resolving these lesions recognizes both DNA and protein, whilst accommodating diverse chemical structures that trap Top2cc.	INTRO
221	227	Top2cc	complex_assembly	The chemical complexity of DNA damage-derived Top2cc necessitates that DNA repair machinery dedicated to resolving these lesions recognizes both DNA and protein, whilst accommodating diverse chemical structures that trap Top2cc.	INTRO
27	30	DNA	chemical	Precisely how the cellular DNA repair machinery navigates these complex lesions is an important aspect of Top2cc repair that has not yet been explored.	INTRO
106	112	Top2cc	complex_assembly	Precisely how the cellular DNA repair machinery navigates these complex lesions is an important aspect of Top2cc repair that has not yet been explored.	INTRO
0	4	Tdp2	protein	Tdp2 processes phosphotyrosyl linkages in diverse DNA damage contexts.	FIG
15	38	phosphotyrosyl linkages	ptm	Tdp2 processes phosphotyrosyl linkages in diverse DNA damage contexts.	FIG
50	53	DNA	chemical	Tdp2 processes phosphotyrosyl linkages in diverse DNA damage contexts.	FIG
15	18	DNA	chemical	(A) Unrepaired DNA damage and repair intermediates such as bulky DNA adducts, ribonucleotides or abasic sites can poison Top2 and trap Top2 cleavage complex (Top2cc), resulting in a DSB with a 5′–Top2 protein adduct linked by a phosphotyrosine bond.	FIG
65	68	DNA	chemical	(A) Unrepaired DNA damage and repair intermediates such as bulky DNA adducts, ribonucleotides or abasic sites can poison Top2 and trap Top2 cleavage complex (Top2cc), resulting in a DSB with a 5′–Top2 protein adduct linked by a phosphotyrosine bond.	FIG
121	125	Top2	protein_type	(A) Unrepaired DNA damage and repair intermediates such as bulky DNA adducts, ribonucleotides or abasic sites can poison Top2 and trap Top2 cleavage complex (Top2cc), resulting in a DSB with a 5′–Top2 protein adduct linked by a phosphotyrosine bond.	FIG
135	139	Top2	protein_type	(A) Unrepaired DNA damage and repair intermediates such as bulky DNA adducts, ribonucleotides or abasic sites can poison Top2 and trap Top2 cleavage complex (Top2cc), resulting in a DSB with a 5′–Top2 protein adduct linked by a phosphotyrosine bond.	FIG
158	164	Top2cc	complex_assembly	(A) Unrepaired DNA damage and repair intermediates such as bulky DNA adducts, ribonucleotides or abasic sites can poison Top2 and trap Top2 cleavage complex (Top2cc), resulting in a DSB with a 5′–Top2 protein adduct linked by a phosphotyrosine bond.	FIG
196	200	Top2	protein_type	(A) Unrepaired DNA damage and repair intermediates such as bulky DNA adducts, ribonucleotides or abasic sites can poison Top2 and trap Top2 cleavage complex (Top2cc), resulting in a DSB with a 5′–Top2 protein adduct linked by a phosphotyrosine bond.	FIG
228	243	phosphotyrosine	residue_name	(A) Unrepaired DNA damage and repair intermediates such as bulky DNA adducts, ribonucleotides or abasic sites can poison Top2 and trap Top2 cleavage complex (Top2cc), resulting in a DSB with a 5′–Top2 protein adduct linked by a phosphotyrosine bond.	FIG
0	4	Tdp2	protein	Tdp2 hydrolyzes the 5′–phosphotyrosine adduct derived from poisoned Top2 leaving DNA ends with a 5′-phosphate, which facilitates DNA end joining through the NHEJ pathway.	FIG
23	38	phosphotyrosine	residue_name	Tdp2 hydrolyzes the 5′–phosphotyrosine adduct derived from poisoned Top2 leaving DNA ends with a 5′-phosphate, which facilitates DNA end joining through the NHEJ pathway.	FIG
68	72	Top2	protein_type	Tdp2 hydrolyzes the 5′–phosphotyrosine adduct derived from poisoned Top2 leaving DNA ends with a 5′-phosphate, which facilitates DNA end joining through the NHEJ pathway.	FIG
81	84	DNA	chemical	Tdp2 hydrolyzes the 5′–phosphotyrosine adduct derived from poisoned Top2 leaving DNA ends with a 5′-phosphate, which facilitates DNA end joining through the NHEJ pathway.	FIG
97	109	5′-phosphate	chemical	Tdp2 hydrolyzes the 5′–phosphotyrosine adduct derived from poisoned Top2 leaving DNA ends with a 5′-phosphate, which facilitates DNA end joining through the NHEJ pathway.	FIG
129	132	DNA	chemical	Tdp2 hydrolyzes the 5′–phosphotyrosine adduct derived from poisoned Top2 leaving DNA ends with a 5′-phosphate, which facilitates DNA end joining through the NHEJ pathway.	FIG
4	7	DNA	chemical	(B) DNA oligonucleotide substrates synthesized by EDC-imidazole coupling and used in Tdp2 enzyme assays contain deoxyadenine (dA), Ethenoadenine (ϵA) or an abasic site (THF) and a 5′–nitrophenol moiety.	FIG
85	103	Tdp2 enzyme assays	experimental_method	(B) DNA oligonucleotide substrates synthesized by EDC-imidazole coupling and used in Tdp2 enzyme assays contain deoxyadenine (dA), Ethenoadenine (ϵA) or an abasic site (THF) and a 5′–nitrophenol moiety.	FIG
112	124	deoxyadenine	chemical	(B) DNA oligonucleotide substrates synthesized by EDC-imidazole coupling and used in Tdp2 enzyme assays contain deoxyadenine (dA), Ethenoadenine (ϵA) or an abasic site (THF) and a 5′–nitrophenol moiety.	FIG
126	128	dA	chemical	(B) DNA oligonucleotide substrates synthesized by EDC-imidazole coupling and used in Tdp2 enzyme assays contain deoxyadenine (dA), Ethenoadenine (ϵA) or an abasic site (THF) and a 5′–nitrophenol moiety.	FIG
131	144	Ethenoadenine	chemical	(B) DNA oligonucleotide substrates synthesized by EDC-imidazole coupling and used in Tdp2 enzyme assays contain deoxyadenine (dA), Ethenoadenine (ϵA) or an abasic site (THF) and a 5′–nitrophenol moiety.	FIG
146	148	ϵA	chemical	(B) DNA oligonucleotide substrates synthesized by EDC-imidazole coupling and used in Tdp2 enzyme assays contain deoxyadenine (dA), Ethenoadenine (ϵA) or an abasic site (THF) and a 5′–nitrophenol moiety.	FIG
156	167	abasic site	site	(B) DNA oligonucleotide substrates synthesized by EDC-imidazole coupling and used in Tdp2 enzyme assays contain deoxyadenine (dA), Ethenoadenine (ϵA) or an abasic site (THF) and a 5′–nitrophenol moiety.	FIG
169	172	THF	chemical	(B) DNA oligonucleotide substrates synthesized by EDC-imidazole coupling and used in Tdp2 enzyme assays contain deoxyadenine (dA), Ethenoadenine (ϵA) or an abasic site (THF) and a 5′–nitrophenol moiety.	FIG
0	14	Phosphotyrosyl	ptm	Phosphotyrosyl bond hydrolysis catalyzed by mTdp2cat releases p-nitrophenol, which is detected by measuring absorbance at 415 nm. (C) mTdp2cat reaction rates on p–nitrophenol modified DNA substrates shown in panel B. Rates are reported as molecules of PNP s−1 produced by mTdp2cat.	FIG
44	52	mTdp2cat	structure_element	Phosphotyrosyl bond hydrolysis catalyzed by mTdp2cat releases p-nitrophenol, which is detected by measuring absorbance at 415 nm. (C) mTdp2cat reaction rates on p–nitrophenol modified DNA substrates shown in panel B. Rates are reported as molecules of PNP s−1 produced by mTdp2cat.	FIG
62	75	p-nitrophenol	chemical	Phosphotyrosyl bond hydrolysis catalyzed by mTdp2cat releases p-nitrophenol, which is detected by measuring absorbance at 415 nm. (C) mTdp2cat reaction rates on p–nitrophenol modified DNA substrates shown in panel B. Rates are reported as molecules of PNP s−1 produced by mTdp2cat.	FIG
134	142	mTdp2cat	structure_element	Phosphotyrosyl bond hydrolysis catalyzed by mTdp2cat releases p-nitrophenol, which is detected by measuring absorbance at 415 nm. (C) mTdp2cat reaction rates on p–nitrophenol modified DNA substrates shown in panel B. Rates are reported as molecules of PNP s−1 produced by mTdp2cat.	FIG
143	157	reaction rates	evidence	Phosphotyrosyl bond hydrolysis catalyzed by mTdp2cat releases p-nitrophenol, which is detected by measuring absorbance at 415 nm. (C) mTdp2cat reaction rates on p–nitrophenol modified DNA substrates shown in panel B. Rates are reported as molecules of PNP s−1 produced by mTdp2cat.	FIG
161	174	p–nitrophenol	chemical	Phosphotyrosyl bond hydrolysis catalyzed by mTdp2cat releases p-nitrophenol, which is detected by measuring absorbance at 415 nm. (C) mTdp2cat reaction rates on p–nitrophenol modified DNA substrates shown in panel B. Rates are reported as molecules of PNP s−1 produced by mTdp2cat.	FIG
184	187	DNA	chemical	Phosphotyrosyl bond hydrolysis catalyzed by mTdp2cat releases p-nitrophenol, which is detected by measuring absorbance at 415 nm. (C) mTdp2cat reaction rates on p–nitrophenol modified DNA substrates shown in panel B. Rates are reported as molecules of PNP s−1 produced by mTdp2cat.	FIG
252	255	PNP	chemical	Phosphotyrosyl bond hydrolysis catalyzed by mTdp2cat releases p-nitrophenol, which is detected by measuring absorbance at 415 nm. (C) mTdp2cat reaction rates on p–nitrophenol modified DNA substrates shown in panel B. Rates are reported as molecules of PNP s−1 produced by mTdp2cat.	FIG
272	280	mTdp2cat	structure_element	Phosphotyrosyl bond hydrolysis catalyzed by mTdp2cat releases p-nitrophenol, which is detected by measuring absorbance at 415 nm. (C) mTdp2cat reaction rates on p–nitrophenol modified DNA substrates shown in panel B. Rates are reported as molecules of PNP s−1 produced by mTdp2cat.	FIG
0	8	P-values	evidence	P-values calculated using two-tailed t-test; error bars, s.d. n = 4, n.s. = not statistically significant. (D) Structure of mTdp2cat bound to 5′-phosphate DNA (product complex) containing ϵA (yellow).	FIG
37	43	t-test	experimental_method	P-values calculated using two-tailed t-test; error bars, s.d. n = 4, n.s. = not statistically significant. (D) Structure of mTdp2cat bound to 5′-phosphate DNA (product complex) containing ϵA (yellow).	FIG
111	120	Structure	evidence	P-values calculated using two-tailed t-test; error bars, s.d. n = 4, n.s. = not statistically significant. (D) Structure of mTdp2cat bound to 5′-phosphate DNA (product complex) containing ϵA (yellow).	FIG
124	132	mTdp2cat	structure_element	P-values calculated using two-tailed t-test; error bars, s.d. n = 4, n.s. = not statistically significant. (D) Structure of mTdp2cat bound to 5′-phosphate DNA (product complex) containing ϵA (yellow).	FIG
133	141	bound to	protein_state	P-values calculated using two-tailed t-test; error bars, s.d. n = 4, n.s. = not statistically significant. (D) Structure of mTdp2cat bound to 5′-phosphate DNA (product complex) containing ϵA (yellow).	FIG
142	158	5′-phosphate DNA	chemical	P-values calculated using two-tailed t-test; error bars, s.d. n = 4, n.s. = not statistically significant. (D) Structure of mTdp2cat bound to 5′-phosphate DNA (product complex) containing ϵA (yellow).	FIG
188	190	ϵA	chemical	P-values calculated using two-tailed t-test; error bars, s.d. n = 4, n.s. = not statistically significant. (D) Structure of mTdp2cat bound to 5′-phosphate DNA (product complex) containing ϵA (yellow).	FIG
0	22	DNA binding β2Hβ–grasp	site	DNA binding β2Hβ–grasp (tan) and cap elements engage the 5′-nucleotide as well as the +2 and +3 nucleotides (blue) of substrate DNA.	FIG
128	131	DNA	chemical	DNA binding β2Hβ–grasp (tan) and cap elements engage the 5′-nucleotide as well as the +2 and +3 nucleotides (blue) of substrate DNA.	FIG
0	22	DNA binding β2Hβ–grasp	site	DNA binding β2Hβ–grasp (tan) and cap elements engage the 5′-nucleotide as well as the +2 and +3 nucleotides (blue) of substrate DNA.	FIG
128	131	DNA	chemical	DNA binding β2Hβ–grasp (tan) and cap elements engage the 5′-nucleotide as well as the +2 and +3 nucleotides (blue) of substrate DNA.	FIG
51	60	Structure	evidence	PDB entry 5HT2 is displayed, also see Table 1. (E) Structure of mTdp2cat bound to 5′-phosphate DNA (product complex) containing THF (yellow).	FIG
64	72	mTdp2cat	structure_element	PDB entry 5HT2 is displayed, also see Table 1. (E) Structure of mTdp2cat bound to 5′-phosphate DNA (product complex) containing THF (yellow).	FIG
73	81	bound to	protein_state	PDB entry 5HT2 is displayed, also see Table 1. (E) Structure of mTdp2cat bound to 5′-phosphate DNA (product complex) containing THF (yellow).	FIG
82	98	5′-phosphate DNA	chemical	PDB entry 5HT2 is displayed, also see Table 1. (E) Structure of mTdp2cat bound to 5′-phosphate DNA (product complex) containing THF (yellow).	FIG
128	131	THF	chemical	PDB entry 5HT2 is displayed, also see Table 1. (E) Structure of mTdp2cat bound to 5′-phosphate DNA (product complex) containing THF (yellow).	FIG
51	60	Structure	evidence	PDB entry 5INK is displayed, also see Table 1. (F) Structure of mTdp2cat in the absence of DNA showing the extended 3-helix loop (tan) open-conformation of the DNA-binding grasp as seen in monomer E of the apo structure.	FIG
64	72	mTdp2cat	structure_element	PDB entry 5INK is displayed, also see Table 1. (F) Structure of mTdp2cat in the absence of DNA showing the extended 3-helix loop (tan) open-conformation of the DNA-binding grasp as seen in monomer E of the apo structure.	FIG
80	90	absence of	protein_state	PDB entry 5INK is displayed, also see Table 1. (F) Structure of mTdp2cat in the absence of DNA showing the extended 3-helix loop (tan) open-conformation of the DNA-binding grasp as seen in monomer E of the apo structure.	FIG
91	94	DNA	chemical	PDB entry 5INK is displayed, also see Table 1. (F) Structure of mTdp2cat in the absence of DNA showing the extended 3-helix loop (tan) open-conformation of the DNA-binding grasp as seen in monomer E of the apo structure.	FIG
107	115	extended	protein_state	PDB entry 5INK is displayed, also see Table 1. (F) Structure of mTdp2cat in the absence of DNA showing the extended 3-helix loop (tan) open-conformation of the DNA-binding grasp as seen in monomer E of the apo structure.	FIG
116	128	3-helix loop	structure_element	PDB entry 5INK is displayed, also see Table 1. (F) Structure of mTdp2cat in the absence of DNA showing the extended 3-helix loop (tan) open-conformation of the DNA-binding grasp as seen in monomer E of the apo structure.	FIG
135	139	open	protein_state	PDB entry 5INK is displayed, also see Table 1. (F) Structure of mTdp2cat in the absence of DNA showing the extended 3-helix loop (tan) open-conformation of the DNA-binding grasp as seen in monomer E of the apo structure.	FIG
160	177	DNA-binding grasp	site	PDB entry 5INK is displayed, also see Table 1. (F) Structure of mTdp2cat in the absence of DNA showing the extended 3-helix loop (tan) open-conformation of the DNA-binding grasp as seen in monomer E of the apo structure.	FIG
189	196	monomer	oligomeric_state	PDB entry 5INK is displayed, also see Table 1. (F) Structure of mTdp2cat in the absence of DNA showing the extended 3-helix loop (tan) open-conformation of the DNA-binding grasp as seen in monomer E of the apo structure.	FIG
197	198	E	structure_element	PDB entry 5INK is displayed, also see Table 1. (F) Structure of mTdp2cat in the absence of DNA showing the extended 3-helix loop (tan) open-conformation of the DNA-binding grasp as seen in monomer E of the apo structure.	FIG
206	209	apo	protein_state	PDB entry 5INK is displayed, also see Table 1. (F) Structure of mTdp2cat in the absence of DNA showing the extended 3-helix loop (tan) open-conformation of the DNA-binding grasp as seen in monomer E of the apo structure.	FIG
210	219	structure	evidence	PDB entry 5INK is displayed, also see Table 1. (F) Structure of mTdp2cat in the absence of DNA showing the extended 3-helix loop (tan) open-conformation of the DNA-binding grasp as seen in monomer E of the apo structure.	FIG
0	31	Tyrosyl DNA phosphodiesterase 2	protein	Tyrosyl DNA phosphodiesterase 2 (Tdp2) directly hydrolyzes 5′-phosphotyrosyl (5′-Y) linkages, and is a key modulator of cellular resistance to chemotherapeutic Top2 poisons.	INTRO
33	37	Tdp2	protein	Tyrosyl DNA phosphodiesterase 2 (Tdp2) directly hydrolyzes 5′-phosphotyrosyl (5′-Y) linkages, and is a key modulator of cellular resistance to chemotherapeutic Top2 poisons.	INTRO
59	76	5′-phosphotyrosyl	ptm	Tyrosyl DNA phosphodiesterase 2 (Tdp2) directly hydrolyzes 5′-phosphotyrosyl (5′-Y) linkages, and is a key modulator of cellular resistance to chemotherapeutic Top2 poisons.	INTRO
78	82	5′-Y	ptm	Tyrosyl DNA phosphodiesterase 2 (Tdp2) directly hydrolyzes 5′-phosphotyrosyl (5′-Y) linkages, and is a key modulator of cellular resistance to chemotherapeutic Top2 poisons.	INTRO
84	92	linkages	ptm	Tyrosyl DNA phosphodiesterase 2 (Tdp2) directly hydrolyzes 5′-phosphotyrosyl (5′-Y) linkages, and is a key modulator of cellular resistance to chemotherapeutic Top2 poisons.	INTRO
160	164	Top2	protein_type	Tyrosyl DNA phosphodiesterase 2 (Tdp2) directly hydrolyzes 5′-phosphotyrosyl (5′-Y) linkages, and is a key modulator of cellular resistance to chemotherapeutic Top2 poisons.	INTRO
0	4	Tdp2	protein	Tdp2 knockdown sensitizes A549 lung cancer cells to etoposide, and increases formation of nuclear γH2AX foci, a marker of DSBs, underlining the importance of Tdp2 in cellular Top2cc repair.	INTRO
5	14	knockdown	experimental_method	Tdp2 knockdown sensitizes A549 lung cancer cells to etoposide, and increases formation of nuclear γH2AX foci, a marker of DSBs, underlining the importance of Tdp2 in cellular Top2cc repair.	INTRO
52	61	etoposide	chemical	Tdp2 knockdown sensitizes A549 lung cancer cells to etoposide, and increases formation of nuclear γH2AX foci, a marker of DSBs, underlining the importance of Tdp2 in cellular Top2cc repair.	INTRO
158	162	Tdp2	protein	Tdp2 knockdown sensitizes A549 lung cancer cells to etoposide, and increases formation of nuclear γH2AX foci, a marker of DSBs, underlining the importance of Tdp2 in cellular Top2cc repair.	INTRO
175	181	Top2cc	complex_assembly	Tdp2 knockdown sensitizes A549 lung cancer cells to etoposide, and increases formation of nuclear γH2AX foci, a marker of DSBs, underlining the importance of Tdp2 in cellular Top2cc repair.	INTRO
0	4	Tdp2	protein	Tdp2 is overexpressed in lung cancers, is transcriptionally up-regulated in mutant p53 cells and mediates mutant p53 gain of function phenotypes, which can lead to acquisition of therapy resistance during cancer progression.	INTRO
76	82	mutant	protein_state	Tdp2 is overexpressed in lung cancers, is transcriptionally up-regulated in mutant p53 cells and mediates mutant p53 gain of function phenotypes, which can lead to acquisition of therapy resistance during cancer progression.	INTRO
83	86	p53	protein	Tdp2 is overexpressed in lung cancers, is transcriptionally up-regulated in mutant p53 cells and mediates mutant p53 gain of function phenotypes, which can lead to acquisition of therapy resistance during cancer progression.	INTRO
106	112	mutant	protein_state	Tdp2 is overexpressed in lung cancers, is transcriptionally up-regulated in mutant p53 cells and mediates mutant p53 gain of function phenotypes, which can lead to acquisition of therapy resistance during cancer progression.	INTRO
113	116	p53	protein	Tdp2 is overexpressed in lung cancers, is transcriptionally up-regulated in mutant p53 cells and mediates mutant p53 gain of function phenotypes, which can lead to acquisition of therapy resistance during cancer progression.	INTRO
18	22	Tdp2	protein	The importance of Tdp2 in mediating topoisomerase biology is further underlined by the facts that human TDP2 inactivating mutations are found in individuals with intellectual disabilities, seizures and ataxia, and at the cellular level, loss of Tdp2 inhibits Top2β-dependent transcription.	INTRO
36	49	topoisomerase	protein_type	The importance of Tdp2 in mediating topoisomerase biology is further underlined by the facts that human TDP2 inactivating mutations are found in individuals with intellectual disabilities, seizures and ataxia, and at the cellular level, loss of Tdp2 inhibits Top2β-dependent transcription.	INTRO
98	103	human	species	The importance of Tdp2 in mediating topoisomerase biology is further underlined by the facts that human TDP2 inactivating mutations are found in individuals with intellectual disabilities, seizures and ataxia, and at the cellular level, loss of Tdp2 inhibits Top2β-dependent transcription.	INTRO
104	108	TDP2	protein	The importance of Tdp2 in mediating topoisomerase biology is further underlined by the facts that human TDP2 inactivating mutations are found in individuals with intellectual disabilities, seizures and ataxia, and at the cellular level, loss of Tdp2 inhibits Top2β-dependent transcription.	INTRO
237	244	loss of	protein_state	The importance of Tdp2 in mediating topoisomerase biology is further underlined by the facts that human TDP2 inactivating mutations are found in individuals with intellectual disabilities, seizures and ataxia, and at the cellular level, loss of Tdp2 inhibits Top2β-dependent transcription.	INTRO
245	249	Tdp2	protein	The importance of Tdp2 in mediating topoisomerase biology is further underlined by the facts that human TDP2 inactivating mutations are found in individuals with intellectual disabilities, seizures and ataxia, and at the cellular level, loss of Tdp2 inhibits Top2β-dependent transcription.	INTRO
259	264	Top2β	protein	The importance of Tdp2 in mediating topoisomerase biology is further underlined by the facts that human TDP2 inactivating mutations are found in individuals with intellectual disabilities, seizures and ataxia, and at the cellular level, loss of Tdp2 inhibits Top2β-dependent transcription.	INTRO
20	24	TDP2	protein	It is possible that TDP2 single nucleotide polymorphisms (SNPs) encode mutations that impact Tdp2 function, but the molecular underpinnings for such Tdp2 deficiencies are not understood.	INTRO
93	97	Tdp2	protein	It is possible that TDP2 single nucleotide polymorphisms (SNPs) encode mutations that impact Tdp2 function, but the molecular underpinnings for such Tdp2 deficiencies are not understood.	INTRO
149	153	Tdp2	protein	It is possible that TDP2 single nucleotide polymorphisms (SNPs) encode mutations that impact Tdp2 function, but the molecular underpinnings for such Tdp2 deficiencies are not understood.	INTRO
39	44	X-ray	experimental_method	Previously we reported high-resolution X-ray crystal structures of the minimal catalytically active endonuclease/exonuclease/phosphatase (EEP) domain of mouse Tdp2 (mTdp2cat) bound to a DNA substrate mimic, and a 5′-phosphorylated reaction product.	INTRO
45	63	crystal structures	evidence	Previously we reported high-resolution X-ray crystal structures of the minimal catalytically active endonuclease/exonuclease/phosphatase (EEP) domain of mouse Tdp2 (mTdp2cat) bound to a DNA substrate mimic, and a 5′-phosphorylated reaction product.	INTRO
71	99	minimal catalytically active	protein_state	Previously we reported high-resolution X-ray crystal structures of the minimal catalytically active endonuclease/exonuclease/phosphatase (EEP) domain of mouse Tdp2 (mTdp2cat) bound to a DNA substrate mimic, and a 5′-phosphorylated reaction product.	INTRO
100	136	endonuclease/exonuclease/phosphatase	structure_element	Previously we reported high-resolution X-ray crystal structures of the minimal catalytically active endonuclease/exonuclease/phosphatase (EEP) domain of mouse Tdp2 (mTdp2cat) bound to a DNA substrate mimic, and a 5′-phosphorylated reaction product.	INTRO
138	141	EEP	structure_element	Previously we reported high-resolution X-ray crystal structures of the minimal catalytically active endonuclease/exonuclease/phosphatase (EEP) domain of mouse Tdp2 (mTdp2cat) bound to a DNA substrate mimic, and a 5′-phosphorylated reaction product.	INTRO
153	158	mouse	taxonomy_domain	Previously we reported high-resolution X-ray crystal structures of the minimal catalytically active endonuclease/exonuclease/phosphatase (EEP) domain of mouse Tdp2 (mTdp2cat) bound to a DNA substrate mimic, and a 5′-phosphorylated reaction product.	INTRO
159	163	Tdp2	protein	Previously we reported high-resolution X-ray crystal structures of the minimal catalytically active endonuclease/exonuclease/phosphatase (EEP) domain of mouse Tdp2 (mTdp2cat) bound to a DNA substrate mimic, and a 5′-phosphorylated reaction product.	INTRO
165	173	mTdp2cat	structure_element	Previously we reported high-resolution X-ray crystal structures of the minimal catalytically active endonuclease/exonuclease/phosphatase (EEP) domain of mouse Tdp2 (mTdp2cat) bound to a DNA substrate mimic, and a 5′-phosphorylated reaction product.	INTRO
175	183	bound to	protein_state	Previously we reported high-resolution X-ray crystal structures of the minimal catalytically active endonuclease/exonuclease/phosphatase (EEP) domain of mouse Tdp2 (mTdp2cat) bound to a DNA substrate mimic, and a 5′-phosphorylated reaction product.	INTRO
186	189	DNA	chemical	Previously we reported high-resolution X-ray crystal structures of the minimal catalytically active endonuclease/exonuclease/phosphatase (EEP) domain of mouse Tdp2 (mTdp2cat) bound to a DNA substrate mimic, and a 5′-phosphorylated reaction product.	INTRO
213	230	5′-phosphorylated	protein_state	Previously we reported high-resolution X-ray crystal structures of the minimal catalytically active endonuclease/exonuclease/phosphatase (EEP) domain of mouse Tdp2 (mTdp2cat) bound to a DNA substrate mimic, and a 5′-phosphorylated reaction product.	INTRO
56	60	Tdp2	protein	However, important questions regarding the mechanism of Tdp2 engagement and processing of DNA damage remain.	INTRO
90	93	DNA	chemical	However, important questions regarding the mechanism of Tdp2 engagement and processing of DNA damage remain.	INTRO
24	28	Tdp2	protein	First, it is unclear if Tdp2 processes phosphotyrosyl linkages in the context of DNA damage that triggers Top2cc, and if so, how the enzyme can accommodate such complex DNA damage within its active site.	INTRO
39	62	phosphotyrosyl linkages	ptm	First, it is unclear if Tdp2 processes phosphotyrosyl linkages in the context of DNA damage that triggers Top2cc, and if so, how the enzyme can accommodate such complex DNA damage within its active site.	INTRO
81	84	DNA	chemical	First, it is unclear if Tdp2 processes phosphotyrosyl linkages in the context of DNA damage that triggers Top2cc, and if so, how the enzyme can accommodate such complex DNA damage within its active site.	INTRO
106	112	Top2cc	complex_assembly	First, it is unclear if Tdp2 processes phosphotyrosyl linkages in the context of DNA damage that triggers Top2cc, and if so, how the enzyme can accommodate such complex DNA damage within its active site.	INTRO
169	172	DNA	chemical	First, it is unclear if Tdp2 processes phosphotyrosyl linkages in the context of DNA damage that triggers Top2cc, and if so, how the enzyme can accommodate such complex DNA damage within its active site.	INTRO
191	202	active site	site	First, it is unclear if Tdp2 processes phosphotyrosyl linkages in the context of DNA damage that triggers Top2cc, and if so, how the enzyme can accommodate such complex DNA damage within its active site.	INTRO
9	20	metal-bound	protein_state	Based on metal-bound Tdp2 structures, we also proposed a single Mg2+ mediated catalytic mechanism, but this mechanism requires further scrutiny and characterization.	INTRO
21	25	Tdp2	protein	Based on metal-bound Tdp2 structures, we also proposed a single Mg2+ mediated catalytic mechanism, but this mechanism requires further scrutiny and characterization.	INTRO
26	36	structures	evidence	Based on metal-bound Tdp2 structures, we also proposed a single Mg2+ mediated catalytic mechanism, but this mechanism requires further scrutiny and characterization.	INTRO
64	68	Mg2+	chemical	Based on metal-bound Tdp2 structures, we also proposed a single Mg2+ mediated catalytic mechanism, but this mechanism requires further scrutiny and characterization.	INTRO
32	56	structure-function study	experimental_method	Herein, we report an integrated structure-function study of the Tdp2 reaction mechanism, including a description of new X-ray structures of ligand-free Tdp2, and Tdp2 bound to abasic and alkylated (1-N6-etheno-adenine) DNA damage.	INTRO
64	68	Tdp2	protein	Herein, we report an integrated structure-function study of the Tdp2 reaction mechanism, including a description of new X-ray structures of ligand-free Tdp2, and Tdp2 bound to abasic and alkylated (1-N6-etheno-adenine) DNA damage.	INTRO
120	125	X-ray	experimental_method	Herein, we report an integrated structure-function study of the Tdp2 reaction mechanism, including a description of new X-ray structures of ligand-free Tdp2, and Tdp2 bound to abasic and alkylated (1-N6-etheno-adenine) DNA damage.	INTRO
126	136	structures	evidence	Herein, we report an integrated structure-function study of the Tdp2 reaction mechanism, including a description of new X-ray structures of ligand-free Tdp2, and Tdp2 bound to abasic and alkylated (1-N6-etheno-adenine) DNA damage.	INTRO
140	151	ligand-free	protein_state	Herein, we report an integrated structure-function study of the Tdp2 reaction mechanism, including a description of new X-ray structures of ligand-free Tdp2, and Tdp2 bound to abasic and alkylated (1-N6-etheno-adenine) DNA damage.	INTRO
152	156	Tdp2	protein	Herein, we report an integrated structure-function study of the Tdp2 reaction mechanism, including a description of new X-ray structures of ligand-free Tdp2, and Tdp2 bound to abasic and alkylated (1-N6-etheno-adenine) DNA damage.	INTRO
162	166	Tdp2	protein	Herein, we report an integrated structure-function study of the Tdp2 reaction mechanism, including a description of new X-ray structures of ligand-free Tdp2, and Tdp2 bound to abasic and alkylated (1-N6-etheno-adenine) DNA damage.	INTRO
167	175	bound to	protein_state	Herein, we report an integrated structure-function study of the Tdp2 reaction mechanism, including a description of new X-ray structures of ligand-free Tdp2, and Tdp2 bound to abasic and alkylated (1-N6-etheno-adenine) DNA damage.	INTRO
198	217	1-N6-etheno-adenine	chemical	Herein, we report an integrated structure-function study of the Tdp2 reaction mechanism, including a description of new X-ray structures of ligand-free Tdp2, and Tdp2 bound to abasic and alkylated (1-N6-etheno-adenine) DNA damage.	INTRO
219	222	DNA	chemical	Herein, we report an integrated structure-function study of the Tdp2 reaction mechanism, including a description of new X-ray structures of ligand-free Tdp2, and Tdp2 bound to abasic and alkylated (1-N6-etheno-adenine) DNA damage.	INTRO
28	47	structural analysis	experimental_method	Our integrated results from structural analysis, mutagenesis, functional assays and quanyum mechanics/molecular mechanics (QM/MM) modeling of the Tdp2 reaction coordinate describe in detail how Tdp2 mediates a single-metal ion tyrosyl DNA phosphodiesterase reaction capable of acting on diverse DNA end damage.	INTRO
49	60	mutagenesis	experimental_method	Our integrated results from structural analysis, mutagenesis, functional assays and quanyum mechanics/molecular mechanics (QM/MM) modeling of the Tdp2 reaction coordinate describe in detail how Tdp2 mediates a single-metal ion tyrosyl DNA phosphodiesterase reaction capable of acting on diverse DNA end damage.	INTRO
62	79	functional assays	experimental_method	Our integrated results from structural analysis, mutagenesis, functional assays and quanyum mechanics/molecular mechanics (QM/MM) modeling of the Tdp2 reaction coordinate describe in detail how Tdp2 mediates a single-metal ion tyrosyl DNA phosphodiesterase reaction capable of acting on diverse DNA end damage.	INTRO
84	121	quanyum mechanics/molecular mechanics	experimental_method	Our integrated results from structural analysis, mutagenesis, functional assays and quanyum mechanics/molecular mechanics (QM/MM) modeling of the Tdp2 reaction coordinate describe in detail how Tdp2 mediates a single-metal ion tyrosyl DNA phosphodiesterase reaction capable of acting on diverse DNA end damage.	INTRO
123	128	QM/MM	experimental_method	Our integrated results from structural analysis, mutagenesis, functional assays and quanyum mechanics/molecular mechanics (QM/MM) modeling of the Tdp2 reaction coordinate describe in detail how Tdp2 mediates a single-metal ion tyrosyl DNA phosphodiesterase reaction capable of acting on diverse DNA end damage.	INTRO
130	138	modeling	experimental_method	Our integrated results from structural analysis, mutagenesis, functional assays and quanyum mechanics/molecular mechanics (QM/MM) modeling of the Tdp2 reaction coordinate describe in detail how Tdp2 mediates a single-metal ion tyrosyl DNA phosphodiesterase reaction capable of acting on diverse DNA end damage.	INTRO
146	150	Tdp2	protein	Our integrated results from structural analysis, mutagenesis, functional assays and quanyum mechanics/molecular mechanics (QM/MM) modeling of the Tdp2 reaction coordinate describe in detail how Tdp2 mediates a single-metal ion tyrosyl DNA phosphodiesterase reaction capable of acting on diverse DNA end damage.	INTRO
194	198	Tdp2	protein	Our integrated results from structural analysis, mutagenesis, functional assays and quanyum mechanics/molecular mechanics (QM/MM) modeling of the Tdp2 reaction coordinate describe in detail how Tdp2 mediates a single-metal ion tyrosyl DNA phosphodiesterase reaction capable of acting on diverse DNA end damage.	INTRO
227	256	tyrosyl DNA phosphodiesterase	protein_type	Our integrated results from structural analysis, mutagenesis, functional assays and quanyum mechanics/molecular mechanics (QM/MM) modeling of the Tdp2 reaction coordinate describe in detail how Tdp2 mediates a single-metal ion tyrosyl DNA phosphodiesterase reaction capable of acting on diverse DNA end damage.	INTRO
295	298	DNA	chemical	Our integrated results from structural analysis, mutagenesis, functional assays and quanyum mechanics/molecular mechanics (QM/MM) modeling of the Tdp2 reaction coordinate describe in detail how Tdp2 mediates a single-metal ion tyrosyl DNA phosphodiesterase reaction capable of acting on diverse DNA end damage.	INTRO
26	29	DNA	chemical	We further establish that DNA damage binding in the Tdp2 active site is linked to conformational change and binding of metal cofactor.	INTRO
52	56	Tdp2	protein	We further establish that DNA damage binding in the Tdp2 active site is linked to conformational change and binding of metal cofactor.	INTRO
57	68	active site	site	We further establish that DNA damage binding in the Tdp2 active site is linked to conformational change and binding of metal cofactor.	INTRO
27	31	Tdp2	protein	Finally, we characterize a Tdp2 SNP that ablates the Tdp2 single metal binding site and Tdp2 substrate induced conformational changes, and confers Top2 drug sensitivity in mammalian cells.	INTRO
41	48	ablates	protein_state	Finally, we characterize a Tdp2 SNP that ablates the Tdp2 single metal binding site and Tdp2 substrate induced conformational changes, and confers Top2 drug sensitivity in mammalian cells.	INTRO
53	57	Tdp2	protein	Finally, we characterize a Tdp2 SNP that ablates the Tdp2 single metal binding site and Tdp2 substrate induced conformational changes, and confers Top2 drug sensitivity in mammalian cells.	INTRO
58	83	single metal binding site	site	Finally, we characterize a Tdp2 SNP that ablates the Tdp2 single metal binding site and Tdp2 substrate induced conformational changes, and confers Top2 drug sensitivity in mammalian cells.	INTRO
88	92	Tdp2	protein	Finally, we characterize a Tdp2 SNP that ablates the Tdp2 single metal binding site and Tdp2 substrate induced conformational changes, and confers Top2 drug sensitivity in mammalian cells.	INTRO
147	151	Top2	protein_type	Finally, we characterize a Tdp2 SNP that ablates the Tdp2 single metal binding site and Tdp2 substrate induced conformational changes, and confers Top2 drug sensitivity in mammalian cells.	INTRO
172	181	mammalian	taxonomy_domain	Finally, we characterize a Tdp2 SNP that ablates the Tdp2 single metal binding site and Tdp2 substrate induced conformational changes, and confers Top2 drug sensitivity in mammalian cells.	INTRO
0	4	Tdp2	protein	Tdp2 processing of compound DNA damage	RESULTS
28	31	DNA	chemical	Tdp2 processing of compound DNA damage	RESULTS
11	15	Top2	protein_type	Two potent Top2 poisons include bulky alkylated DNA helix-distorting DNA base adducts (e.g. 1-N6-ethenoadenine, ϵA) and abundant abasic sites (Figure 1A).	RESULTS
48	51	DNA	chemical	Two potent Top2 poisons include bulky alkylated DNA helix-distorting DNA base adducts (e.g. 1-N6-ethenoadenine, ϵA) and abundant abasic sites (Figure 1A).	RESULTS
69	72	DNA	chemical	Two potent Top2 poisons include bulky alkylated DNA helix-distorting DNA base adducts (e.g. 1-N6-ethenoadenine, ϵA) and abundant abasic sites (Figure 1A).	RESULTS
92	110	1-N6-ethenoadenine	chemical	Two potent Top2 poisons include bulky alkylated DNA helix-distorting DNA base adducts (e.g. 1-N6-ethenoadenine, ϵA) and abundant abasic sites (Figure 1A).	RESULTS
112	114	ϵA	chemical	Two potent Top2 poisons include bulky alkylated DNA helix-distorting DNA base adducts (e.g. 1-N6-ethenoadenine, ϵA) and abundant abasic sites (Figure 1A).	RESULTS
8	12	Tdp2	protein	Whether Tdp2 processes phosphotyrosyl linkages within these diverse structural contexts is not known.	RESULTS
23	46	phosphotyrosyl linkages	ptm	Whether Tdp2 processes phosphotyrosyl linkages within these diverse structural contexts is not known.	RESULTS
28	47	EDC coupling method	experimental_method	To test this, we adapted an EDC coupling method to generate 5′-terminal p-nitrophenol (PNP) modified oligonucleotides that also harbored DNA damage at the 5′-nucleotide position (see Materials and Methods).	RESULTS
72	85	p-nitrophenol	chemical	To test this, we adapted an EDC coupling method to generate 5′-terminal p-nitrophenol (PNP) modified oligonucleotides that also harbored DNA damage at the 5′-nucleotide position (see Materials and Methods).	RESULTS
87	90	PNP	chemical	To test this, we adapted an EDC coupling method to generate 5′-terminal p-nitrophenol (PNP) modified oligonucleotides that also harbored DNA damage at the 5′-nucleotide position (see Materials and Methods).	RESULTS
137	140	DNA	chemical	To test this, we adapted an EDC coupling method to generate 5′-terminal p-nitrophenol (PNP) modified oligonucleotides that also harbored DNA damage at the 5′-nucleotide position (see Materials and Methods).	RESULTS
56	61	mouse	taxonomy_domain	We then evaluated the ability of a recombinant purified mouse Tdp2 catalytic domain (mTdp2cat) to release PNP (a structural mimic of a topoisomerase tyrosine) from the 5′-terminus of compound damaged DNA substrates using a colorimetric assay (Figure 1B).	RESULTS
62	66	Tdp2	protein	We then evaluated the ability of a recombinant purified mouse Tdp2 catalytic domain (mTdp2cat) to release PNP (a structural mimic of a topoisomerase tyrosine) from the 5′-terminus of compound damaged DNA substrates using a colorimetric assay (Figure 1B).	RESULTS
67	83	catalytic domain	structure_element	We then evaluated the ability of a recombinant purified mouse Tdp2 catalytic domain (mTdp2cat) to release PNP (a structural mimic of a topoisomerase tyrosine) from the 5′-terminus of compound damaged DNA substrates using a colorimetric assay (Figure 1B).	RESULTS
85	93	mTdp2cat	structure_element	We then evaluated the ability of a recombinant purified mouse Tdp2 catalytic domain (mTdp2cat) to release PNP (a structural mimic of a topoisomerase tyrosine) from the 5′-terminus of compound damaged DNA substrates using a colorimetric assay (Figure 1B).	RESULTS
106	109	PNP	chemical	We then evaluated the ability of a recombinant purified mouse Tdp2 catalytic domain (mTdp2cat) to release PNP (a structural mimic of a topoisomerase tyrosine) from the 5′-terminus of compound damaged DNA substrates using a colorimetric assay (Figure 1B).	RESULTS
135	148	topoisomerase	protein_type	We then evaluated the ability of a recombinant purified mouse Tdp2 catalytic domain (mTdp2cat) to release PNP (a structural mimic of a topoisomerase tyrosine) from the 5′-terminus of compound damaged DNA substrates using a colorimetric assay (Figure 1B).	RESULTS
149	157	tyrosine	residue_name	We then evaluated the ability of a recombinant purified mouse Tdp2 catalytic domain (mTdp2cat) to release PNP (a structural mimic of a topoisomerase tyrosine) from the 5′-terminus of compound damaged DNA substrates using a colorimetric assay (Figure 1B).	RESULTS
200	203	DNA	chemical	We then evaluated the ability of a recombinant purified mouse Tdp2 catalytic domain (mTdp2cat) to release PNP (a structural mimic of a topoisomerase tyrosine) from the 5′-terminus of compound damaged DNA substrates using a colorimetric assay (Figure 1B).	RESULTS
223	241	colorimetric assay	experimental_method	We then evaluated the ability of a recombinant purified mouse Tdp2 catalytic domain (mTdp2cat) to release PNP (a structural mimic of a topoisomerase tyrosine) from the 5′-terminus of compound damaged DNA substrates using a colorimetric assay (Figure 1B).	RESULTS
18	22	Tdp2	protein	We observe robust Tdp2-dependent release of PNP from 5′-modified oligonucleotides in the context of dA-PNP, ϵA-PNP or the abasic-site analog tetrahydrofuran spacer (THF) (Figure 1C).	RESULTS
44	47	PNP	chemical	We observe robust Tdp2-dependent release of PNP from 5′-modified oligonucleotides in the context of dA-PNP, ϵA-PNP or the abasic-site analog tetrahydrofuran spacer (THF) (Figure 1C).	RESULTS
100	106	dA-PNP	chemical	We observe robust Tdp2-dependent release of PNP from 5′-modified oligonucleotides in the context of dA-PNP, ϵA-PNP or the abasic-site analog tetrahydrofuran spacer (THF) (Figure 1C).	RESULTS
108	114	ϵA-PNP	chemical	We observe robust Tdp2-dependent release of PNP from 5′-modified oligonucleotides in the context of dA-PNP, ϵA-PNP or the abasic-site analog tetrahydrofuran spacer (THF) (Figure 1C).	RESULTS
141	163	tetrahydrofuran spacer	chemical	We observe robust Tdp2-dependent release of PNP from 5′-modified oligonucleotides in the context of dA-PNP, ϵA-PNP or the abasic-site analog tetrahydrofuran spacer (THF) (Figure 1C).	RESULTS
165	168	THF	chemical	We observe robust Tdp2-dependent release of PNP from 5′-modified oligonucleotides in the context of dA-PNP, ϵA-PNP or the abasic-site analog tetrahydrofuran spacer (THF) (Figure 1C).	RESULTS
6	10	Tdp2	protein	Thus, Tdp2 efficiently cleaves phosphotyrosyl linkages in the context of a compound 5′ lesions composed of abasic or bulky DNA base adduct DNA damage.	RESULTS
31	54	phosphotyrosyl linkages	ptm	Thus, Tdp2 efficiently cleaves phosphotyrosyl linkages in the context of a compound 5′ lesions composed of abasic or bulky DNA base adduct DNA damage.	RESULTS
123	126	DNA	chemical	Thus, Tdp2 efficiently cleaves phosphotyrosyl linkages in the context of a compound 5′ lesions composed of abasic or bulky DNA base adduct DNA damage.	RESULTS
139	142	DNA	chemical	Thus, Tdp2 efficiently cleaves phosphotyrosyl linkages in the context of a compound 5′ lesions composed of abasic or bulky DNA base adduct DNA damage.	RESULTS
38	42	Tdp2	protein	To understand the molecular basis for Tdp2 processing of Top2cc in the context of DNA damage, we crystallized and determined X-ray crystal structures of mTdp2cat bound to 5′-phosphate DNA (product complex) with a 5′-ϵA at 1.43 Å resolution (PDB entry 5HT2) and the abasic DNA damage mimic 5′-THF at 2.15 Å resolution (PDB entry 5INK; Figure 1D and E, Table 1).	RESULTS
57	63	Top2cc	complex_assembly	To understand the molecular basis for Tdp2 processing of Top2cc in the context of DNA damage, we crystallized and determined X-ray crystal structures of mTdp2cat bound to 5′-phosphate DNA (product complex) with a 5′-ϵA at 1.43 Å resolution (PDB entry 5HT2) and the abasic DNA damage mimic 5′-THF at 2.15 Å resolution (PDB entry 5INK; Figure 1D and E, Table 1).	RESULTS
82	85	DNA	chemical	To understand the molecular basis for Tdp2 processing of Top2cc in the context of DNA damage, we crystallized and determined X-ray crystal structures of mTdp2cat bound to 5′-phosphate DNA (product complex) with a 5′-ϵA at 1.43 Å resolution (PDB entry 5HT2) and the abasic DNA damage mimic 5′-THF at 2.15 Å resolution (PDB entry 5INK; Figure 1D and E, Table 1).	RESULTS
97	124	crystallized and determined	experimental_method	To understand the molecular basis for Tdp2 processing of Top2cc in the context of DNA damage, we crystallized and determined X-ray crystal structures of mTdp2cat bound to 5′-phosphate DNA (product complex) with a 5′-ϵA at 1.43 Å resolution (PDB entry 5HT2) and the abasic DNA damage mimic 5′-THF at 2.15 Å resolution (PDB entry 5INK; Figure 1D and E, Table 1).	RESULTS
125	130	X-ray	experimental_method	To understand the molecular basis for Tdp2 processing of Top2cc in the context of DNA damage, we crystallized and determined X-ray crystal structures of mTdp2cat bound to 5′-phosphate DNA (product complex) with a 5′-ϵA at 1.43 Å resolution (PDB entry 5HT2) and the abasic DNA damage mimic 5′-THF at 2.15 Å resolution (PDB entry 5INK; Figure 1D and E, Table 1).	RESULTS
131	149	crystal structures	evidence	To understand the molecular basis for Tdp2 processing of Top2cc in the context of DNA damage, we crystallized and determined X-ray crystal structures of mTdp2cat bound to 5′-phosphate DNA (product complex) with a 5′-ϵA at 1.43 Å resolution (PDB entry 5HT2) and the abasic DNA damage mimic 5′-THF at 2.15 Å resolution (PDB entry 5INK; Figure 1D and E, Table 1).	RESULTS
153	161	mTdp2cat	structure_element	To understand the molecular basis for Tdp2 processing of Top2cc in the context of DNA damage, we crystallized and determined X-ray crystal structures of mTdp2cat bound to 5′-phosphate DNA (product complex) with a 5′-ϵA at 1.43 Å resolution (PDB entry 5HT2) and the abasic DNA damage mimic 5′-THF at 2.15 Å resolution (PDB entry 5INK; Figure 1D and E, Table 1).	RESULTS
162	170	bound to	protein_state	To understand the molecular basis for Tdp2 processing of Top2cc in the context of DNA damage, we crystallized and determined X-ray crystal structures of mTdp2cat bound to 5′-phosphate DNA (product complex) with a 5′-ϵA at 1.43 Å resolution (PDB entry 5HT2) and the abasic DNA damage mimic 5′-THF at 2.15 Å resolution (PDB entry 5INK; Figure 1D and E, Table 1).	RESULTS
171	187	5′-phosphate DNA	chemical	To understand the molecular basis for Tdp2 processing of Top2cc in the context of DNA damage, we crystallized and determined X-ray crystal structures of mTdp2cat bound to 5′-phosphate DNA (product complex) with a 5′-ϵA at 1.43 Å resolution (PDB entry 5HT2) and the abasic DNA damage mimic 5′-THF at 2.15 Å resolution (PDB entry 5INK; Figure 1D and E, Table 1).	RESULTS
213	218	5′-ϵA	chemical	To understand the molecular basis for Tdp2 processing of Top2cc in the context of DNA damage, we crystallized and determined X-ray crystal structures of mTdp2cat bound to 5′-phosphate DNA (product complex) with a 5′-ϵA at 1.43 Å resolution (PDB entry 5HT2) and the abasic DNA damage mimic 5′-THF at 2.15 Å resolution (PDB entry 5INK; Figure 1D and E, Table 1).	RESULTS
272	275	DNA	chemical	To understand the molecular basis for Tdp2 processing of Top2cc in the context of DNA damage, we crystallized and determined X-ray crystal structures of mTdp2cat bound to 5′-phosphate DNA (product complex) with a 5′-ϵA at 1.43 Å resolution (PDB entry 5HT2) and the abasic DNA damage mimic 5′-THF at 2.15 Å resolution (PDB entry 5INK; Figure 1D and E, Table 1).	RESULTS
289	295	5′-THF	chemical	To understand the molecular basis for Tdp2 processing of Top2cc in the context of DNA damage, we crystallized and determined X-ray crystal structures of mTdp2cat bound to 5′-phosphate DNA (product complex) with a 5′-ϵA at 1.43 Å resolution (PDB entry 5HT2) and the abasic DNA damage mimic 5′-THF at 2.15 Å resolution (PDB entry 5INK; Figure 1D and E, Table 1).	RESULTS
9	17	Tdp2-DNA	complex_assembly	In these Tdp2-DNA complex structures, mTdp2cat adopts a mixed α-β fold typified by a central 12-stranded anti-parallel β-sandwich enveloped by several helical elements that mold the Tdp2 active site.	RESULTS
26	36	structures	evidence	In these Tdp2-DNA complex structures, mTdp2cat adopts a mixed α-β fold typified by a central 12-stranded anti-parallel β-sandwich enveloped by several helical elements that mold the Tdp2 active site.	RESULTS
38	46	mTdp2cat	structure_element	In these Tdp2-DNA complex structures, mTdp2cat adopts a mixed α-β fold typified by a central 12-stranded anti-parallel β-sandwich enveloped by several helical elements that mold the Tdp2 active site.	RESULTS
56	70	mixed α-β fold	structure_element	In these Tdp2-DNA complex structures, mTdp2cat adopts a mixed α-β fold typified by a central 12-stranded anti-parallel β-sandwich enveloped by several helical elements that mold the Tdp2 active site.	RESULTS
93	129	12-stranded anti-parallel β-sandwich	structure_element	In these Tdp2-DNA complex structures, mTdp2cat adopts a mixed α-β fold typified by a central 12-stranded anti-parallel β-sandwich enveloped by several helical elements that mold the Tdp2 active site.	RESULTS
182	186	Tdp2	protein	In these Tdp2-DNA complex structures, mTdp2cat adopts a mixed α-β fold typified by a central 12-stranded anti-parallel β-sandwich enveloped by several helical elements that mold the Tdp2 active site.	RESULTS
187	198	active site	site	In these Tdp2-DNA complex structures, mTdp2cat adopts a mixed α-β fold typified by a central 12-stranded anti-parallel β-sandwich enveloped by several helical elements that mold the Tdp2 active site.	RESULTS
70	87	DNA-binding cleft	site	One half of the molecule contributes to formation of the walls of the DNA-binding cleft that embraces the terminal position of the damaged DNA substrate.	RESULTS
139	142	DNA	chemical	One half of the molecule contributes to formation of the walls of the DNA-binding cleft that embraces the terminal position of the damaged DNA substrate.	RESULTS
7	23	DNA lesion-bound	protein_state	In the DNA lesion-bound state, two key DNA binding elements, the β-2-helix-β (β2Hβ) ‘grasp’, and ‘helical cap’ mold the substrate binding trench and direct the ssDNA of a 5′-overhang substrate into the active site.	RESULTS
39	42	DNA	chemical	In the DNA lesion-bound state, two key DNA binding elements, the β-2-helix-β (β2Hβ) ‘grasp’, and ‘helical cap’ mold the substrate binding trench and direct the ssDNA of a 5′-overhang substrate into the active site.	RESULTS
65	76	β-2-helix-β	structure_element	In the DNA lesion-bound state, two key DNA binding elements, the β-2-helix-β (β2Hβ) ‘grasp’, and ‘helical cap’ mold the substrate binding trench and direct the ssDNA of a 5′-overhang substrate into the active site.	RESULTS
78	82	β2Hβ	structure_element	In the DNA lesion-bound state, two key DNA binding elements, the β-2-helix-β (β2Hβ) ‘grasp’, and ‘helical cap’ mold the substrate binding trench and direct the ssDNA of a 5′-overhang substrate into the active site.	RESULTS
85	90	grasp	structure_element	In the DNA lesion-bound state, two key DNA binding elements, the β-2-helix-β (β2Hβ) ‘grasp’, and ‘helical cap’ mold the substrate binding trench and direct the ssDNA of a 5′-overhang substrate into the active site.	RESULTS
98	109	helical cap	structure_element	In the DNA lesion-bound state, two key DNA binding elements, the β-2-helix-β (β2Hβ) ‘grasp’, and ‘helical cap’ mold the substrate binding trench and direct the ssDNA of a 5′-overhang substrate into the active site.	RESULTS
120	144	substrate binding trench	site	In the DNA lesion-bound state, two key DNA binding elements, the β-2-helix-β (β2Hβ) ‘grasp’, and ‘helical cap’ mold the substrate binding trench and direct the ssDNA of a 5′-overhang substrate into the active site.	RESULTS
160	165	ssDNA	chemical	In the DNA lesion-bound state, two key DNA binding elements, the β-2-helix-β (β2Hβ) ‘grasp’, and ‘helical cap’ mold the substrate binding trench and direct the ssDNA of a 5′-overhang substrate into the active site.	RESULTS
202	213	active site	site	In the DNA lesion-bound state, two key DNA binding elements, the β-2-helix-β (β2Hβ) ‘grasp’, and ‘helical cap’ mold the substrate binding trench and direct the ssDNA of a 5′-overhang substrate into the active site.	RESULTS
34	43	structure	evidence	A comparison to an additional new structure of DNA-free Tdp2 (apo state, Figure 1F) shows that this loop is conformationally mobile and important for engaging DNA substrates.	RESULTS
47	55	DNA-free	protein_state	A comparison to an additional new structure of DNA-free Tdp2 (apo state, Figure 1F) shows that this loop is conformationally mobile and important for engaging DNA substrates.	RESULTS
56	60	Tdp2	protein	A comparison to an additional new structure of DNA-free Tdp2 (apo state, Figure 1F) shows that this loop is conformationally mobile and important for engaging DNA substrates.	RESULTS
62	65	apo	protein_state	A comparison to an additional new structure of DNA-free Tdp2 (apo state, Figure 1F) shows that this loop is conformationally mobile and important for engaging DNA substrates.	RESULTS
100	104	loop	structure_element	A comparison to an additional new structure of DNA-free Tdp2 (apo state, Figure 1F) shows that this loop is conformationally mobile and important for engaging DNA substrates.	RESULTS
108	131	conformationally mobile	protein_state	A comparison to an additional new structure of DNA-free Tdp2 (apo state, Figure 1F) shows that this loop is conformationally mobile and important for engaging DNA substrates.	RESULTS
159	162	DNA	chemical	A comparison to an additional new structure of DNA-free Tdp2 (apo state, Figure 1F) shows that this loop is conformationally mobile and important for engaging DNA substrates.	RESULTS
57	59	ϵA	chemical	The mode of engagement of the 5′-nucleobase of the bulky ϵA adduct describes a mechanism for Tdp2 to bind 5′-tyrosylated substrates that contain diverse forms of DNA damage.	RESULTS
93	97	Tdp2	protein	The mode of engagement of the 5′-nucleobase of the bulky ϵA adduct describes a mechanism for Tdp2 to bind 5′-tyrosylated substrates that contain diverse forms of DNA damage.	RESULTS
106	120	5′-tyrosylated	protein_state	The mode of engagement of the 5′-nucleobase of the bulky ϵA adduct describes a mechanism for Tdp2 to bind 5′-tyrosylated substrates that contain diverse forms of DNA damage.	RESULTS
162	165	DNA	chemical	The mode of engagement of the 5′-nucleobase of the bulky ϵA adduct describes a mechanism for Tdp2 to bind 5′-tyrosylated substrates that contain diverse forms of DNA damage.	RESULTS
4	9	5′-ϵA	chemical	The 5′-ϵA nucleobase is recognized by an extended Tdp2 van Der Waals interaction surface, referred to here as the ‘hydrophobic wall’ that is assembled with the sidechains of residues Leu315 and Ile317 (Figure 2A and B).	RESULTS
50	54	Tdp2	protein	The 5′-ϵA nucleobase is recognized by an extended Tdp2 van Der Waals interaction surface, referred to here as the ‘hydrophobic wall’ that is assembled with the sidechains of residues Leu315 and Ile317 (Figure 2A and B).	RESULTS
55	88	van Der Waals interaction surface	site	The 5′-ϵA nucleobase is recognized by an extended Tdp2 van Der Waals interaction surface, referred to here as the ‘hydrophobic wall’ that is assembled with the sidechains of residues Leu315 and Ile317 (Figure 2A and B).	RESULTS
115	131	hydrophobic wall	site	The 5′-ϵA nucleobase is recognized by an extended Tdp2 van Der Waals interaction surface, referred to here as the ‘hydrophobic wall’ that is assembled with the sidechains of residues Leu315 and Ile317 (Figure 2A and B).	RESULTS
183	189	Leu315	residue_name_number	The 5′-ϵA nucleobase is recognized by an extended Tdp2 van Der Waals interaction surface, referred to here as the ‘hydrophobic wall’ that is assembled with the sidechains of residues Leu315 and Ile317 (Figure 2A and B).	RESULTS
194	200	Ile317	residue_name_number	The 5′-ϵA nucleobase is recognized by an extended Tdp2 van Der Waals interaction surface, referred to here as the ‘hydrophobic wall’ that is assembled with the sidechains of residues Leu315 and Ile317 (Figure 2A and B).	RESULTS
0	10	Structures	evidence	Structures of mTdp2cat bound to DNA damage that triggers Top2 poisoning.	FIG
14	22	mTdp2cat	structure_element	Structures of mTdp2cat bound to DNA damage that triggers Top2 poisoning.	FIG
23	31	bound to	protein_state	Structures of mTdp2cat bound to DNA damage that triggers Top2 poisoning.	FIG
32	35	DNA	chemical	Structures of mTdp2cat bound to DNA damage that triggers Top2 poisoning.	FIG
57	61	Top2	protein_type	Structures of mTdp2cat bound to DNA damage that triggers Top2 poisoning.	FIG
4	13	Structure	evidence	(A) Structure of mTdp2cat bound to 5′-phosphate DNA (product complex) containing ϵA (yellow), Mg2+ (magenta) and its inner-sphere waters (gray).	FIG
17	25	mTdp2cat	structure_element	(A) Structure of mTdp2cat bound to 5′-phosphate DNA (product complex) containing ϵA (yellow), Mg2+ (magenta) and its inner-sphere waters (gray).	FIG
26	34	bound to	protein_state	(A) Structure of mTdp2cat bound to 5′-phosphate DNA (product complex) containing ϵA (yellow), Mg2+ (magenta) and its inner-sphere waters (gray).	FIG
35	51	5′-phosphate DNA	chemical	(A) Structure of mTdp2cat bound to 5′-phosphate DNA (product complex) containing ϵA (yellow), Mg2+ (magenta) and its inner-sphere waters (gray).	FIG
81	83	ϵA	chemical	(A) Structure of mTdp2cat bound to 5′-phosphate DNA (product complex) containing ϵA (yellow), Mg2+ (magenta) and its inner-sphere waters (gray).	FIG
94	98	Mg2+	chemical	(A) Structure of mTdp2cat bound to 5′-phosphate DNA (product complex) containing ϵA (yellow), Mg2+ (magenta) and its inner-sphere waters (gray).	FIG
130	136	waters	chemical	(A) Structure of mTdp2cat bound to 5′-phosphate DNA (product complex) containing ϵA (yellow), Mg2+ (magenta) and its inner-sphere waters (gray).	FIG
0	8	mTdp2cat	structure_element	mTdp2cat is colored by electrostatic surface potential (red = negative, blue = positive, gray = neutral/hydrophobic).	FIG
4	44	σ-A weighted 2Fo-Fc electron density map	evidence	(B) σ-A weighted 2Fo-Fc electron density map (at 1.43 Å resolution, contoured at 2.0 σ) for the ϵA DNA complex.	FIG
96	102	ϵA DNA	chemical	(B) σ-A weighted 2Fo-Fc electron density map (at 1.43 Å resolution, contoured at 2.0 σ) for the ϵA DNA complex.	FIG
4	6	ϵA	chemical	The ϵA nucleotide is shown in yellow and a hydrogen bond from the ϵA O4′ to inner-sphere water is shown as gray dashes. (C) Structure of mTdp2cat bound to 5′-phosphate DNA (product complex) containing THF (yellow), Mg2+ (magenta) and its inner-sphere waters (gray).	FIG
43	56	hydrogen bond	bond_interaction	The ϵA nucleotide is shown in yellow and a hydrogen bond from the ϵA O4′ to inner-sphere water is shown as gray dashes. (C) Structure of mTdp2cat bound to 5′-phosphate DNA (product complex) containing THF (yellow), Mg2+ (magenta) and its inner-sphere waters (gray).	FIG
66	68	ϵA	chemical	The ϵA nucleotide is shown in yellow and a hydrogen bond from the ϵA O4′ to inner-sphere water is shown as gray dashes. (C) Structure of mTdp2cat bound to 5′-phosphate DNA (product complex) containing THF (yellow), Mg2+ (magenta) and its inner-sphere waters (gray).	FIG
89	94	water	chemical	The ϵA nucleotide is shown in yellow and a hydrogen bond from the ϵA O4′ to inner-sphere water is shown as gray dashes. (C) Structure of mTdp2cat bound to 5′-phosphate DNA (product complex) containing THF (yellow), Mg2+ (magenta) and its inner-sphere waters (gray).	FIG
124	133	Structure	evidence	The ϵA nucleotide is shown in yellow and a hydrogen bond from the ϵA O4′ to inner-sphere water is shown as gray dashes. (C) Structure of mTdp2cat bound to 5′-phosphate DNA (product complex) containing THF (yellow), Mg2+ (magenta) and its inner-sphere waters (gray).	FIG
137	145	mTdp2cat	structure_element	The ϵA nucleotide is shown in yellow and a hydrogen bond from the ϵA O4′ to inner-sphere water is shown as gray dashes. (C) Structure of mTdp2cat bound to 5′-phosphate DNA (product complex) containing THF (yellow), Mg2+ (magenta) and its inner-sphere waters (gray).	FIG
146	154	bound to	protein_state	The ϵA nucleotide is shown in yellow and a hydrogen bond from the ϵA O4′ to inner-sphere water is shown as gray dashes. (C) Structure of mTdp2cat bound to 5′-phosphate DNA (product complex) containing THF (yellow), Mg2+ (magenta) and its inner-sphere waters (gray).	FIG
155	171	5′-phosphate DNA	chemical	The ϵA nucleotide is shown in yellow and a hydrogen bond from the ϵA O4′ to inner-sphere water is shown as gray dashes. (C) Structure of mTdp2cat bound to 5′-phosphate DNA (product complex) containing THF (yellow), Mg2+ (magenta) and its inner-sphere waters (gray).	FIG
201	204	THF	chemical	The ϵA nucleotide is shown in yellow and a hydrogen bond from the ϵA O4′ to inner-sphere water is shown as gray dashes. (C) Structure of mTdp2cat bound to 5′-phosphate DNA (product complex) containing THF (yellow), Mg2+ (magenta) and its inner-sphere waters (gray).	FIG
215	219	Mg2+	chemical	The ϵA nucleotide is shown in yellow and a hydrogen bond from the ϵA O4′ to inner-sphere water is shown as gray dashes. (C) Structure of mTdp2cat bound to 5′-phosphate DNA (product complex) containing THF (yellow), Mg2+ (magenta) and its inner-sphere waters (gray).	FIG
251	257	waters	chemical	The ϵA nucleotide is shown in yellow and a hydrogen bond from the ϵA O4′ to inner-sphere water is shown as gray dashes. (C) Structure of mTdp2cat bound to 5′-phosphate DNA (product complex) containing THF (yellow), Mg2+ (magenta) and its inner-sphere waters (gray).	FIG
0	8	mTdp2cat	structure_element	mTdp2cat is colored with red (electronegative), blue (electropositive) and gray (hydrophobic) electrostatic surface potential displayed.	FIG
33	73	σ-A weighted 2Fo-Fc electron density map	evidence	PDB entry 5INK is displayed. (D) σ-A weighted 2Fo-Fc electron density map (at 2.15 Å resolution, contoured at 2.0 σ) for THF-DNA complex.	FIG
121	128	THF-DNA	complex_assembly	PDB entry 5INK is displayed. (D) σ-A weighted 2Fo-Fc electron density map (at 2.15 Å resolution, contoured at 2.0 σ) for THF-DNA complex.	FIG
4	7	THF	chemical	The THF is shown in yellow and a hydrogen bond from the THF O4′ to inner-sphere water is shown as gray dashes.	FIG
33	46	hydrogen bond	bond_interaction	The THF is shown in yellow and a hydrogen bond from the THF O4′ to inner-sphere water is shown as gray dashes.	FIG
56	59	THF	chemical	The THF is shown in yellow and a hydrogen bond from the THF O4′ to inner-sphere water is shown as gray dashes.	FIG
80	85	water	chemical	The THF is shown in yellow and a hydrogen bond from the THF O4′ to inner-sphere water is shown as gray dashes.	FIG
24	34	determined	experimental_method	For comparison, we also determined a structure of an undamaged 5′-adenine (5′-dA) bound to Tdp2 at 1.55 Å (PDB entry 5INL).	RESULTS
37	46	structure	evidence	For comparison, we also determined a structure of an undamaged 5′-adenine (5′-dA) bound to Tdp2 at 1.55 Å (PDB entry 5INL).	RESULTS
63	73	5′-adenine	chemical	For comparison, we also determined a structure of an undamaged 5′-adenine (5′-dA) bound to Tdp2 at 1.55 Å (PDB entry 5INL).	RESULTS
75	80	5′-dA	chemical	For comparison, we also determined a structure of an undamaged 5′-adenine (5′-dA) bound to Tdp2 at 1.55 Å (PDB entry 5INL).	RESULTS
82	90	bound to	protein_state	For comparison, we also determined a structure of an undamaged 5′-adenine (5′-dA) bound to Tdp2 at 1.55 Å (PDB entry 5INL).	RESULTS
91	95	Tdp2	protein	For comparison, we also determined a structure of an undamaged 5′-adenine (5′-dA) bound to Tdp2 at 1.55 Å (PDB entry 5INL).	RESULTS
2	20	structural overlay	experimental_method	A structural overlay of damaged and undamaged nucleotides shows no major distortions to nucleotide planarity between different bound sequences and DNA damage (compare ϵA, dA and dC, Supplementary Figure S1A–D).	RESULTS
127	132	bound	protein_state	A structural overlay of damaged and undamaged nucleotides shows no major distortions to nucleotide planarity between different bound sequences and DNA damage (compare ϵA, dA and dC, Supplementary Figure S1A–D).	RESULTS
147	150	DNA	chemical	A structural overlay of damaged and undamaged nucleotides shows no major distortions to nucleotide planarity between different bound sequences and DNA damage (compare ϵA, dA and dC, Supplementary Figure S1A–D).	RESULTS
167	169	ϵA	chemical	A structural overlay of damaged and undamaged nucleotides shows no major distortions to nucleotide planarity between different bound sequences and DNA damage (compare ϵA, dA and dC, Supplementary Figure S1A–D).	RESULTS
171	173	dA	chemical	A structural overlay of damaged and undamaged nucleotides shows no major distortions to nucleotide planarity between different bound sequences and DNA damage (compare ϵA, dA and dC, Supplementary Figure S1A–D).	RESULTS
178	180	dC	chemical	A structural overlay of damaged and undamaged nucleotides shows no major distortions to nucleotide planarity between different bound sequences and DNA damage (compare ϵA, dA and dC, Supplementary Figure S1A–D).	RESULTS
67	69	ϵG	chemical	Therefore, structurally diverse undamaged or alkylated bases (e.g. ϵG, ϵT) could likely be accommodated in the Tdp2 active site via planar base stacking with the active site facing hydrophobic wall of the β2Hβ motif.	RESULTS
71	73	ϵT	chemical	Therefore, structurally diverse undamaged or alkylated bases (e.g. ϵG, ϵT) could likely be accommodated in the Tdp2 active site via planar base stacking with the active site facing hydrophobic wall of the β2Hβ motif.	RESULTS
111	115	Tdp2	protein	Therefore, structurally diverse undamaged or alkylated bases (e.g. ϵG, ϵT) could likely be accommodated in the Tdp2 active site via planar base stacking with the active site facing hydrophobic wall of the β2Hβ motif.	RESULTS
116	127	active site	site	Therefore, structurally diverse undamaged or alkylated bases (e.g. ϵG, ϵT) could likely be accommodated in the Tdp2 active site via planar base stacking with the active site facing hydrophobic wall of the β2Hβ motif.	RESULTS
132	152	planar base stacking	bond_interaction	Therefore, structurally diverse undamaged or alkylated bases (e.g. ϵG, ϵT) could likely be accommodated in the Tdp2 active site via planar base stacking with the active site facing hydrophobic wall of the β2Hβ motif.	RESULTS
162	173	active site	site	Therefore, structurally diverse undamaged or alkylated bases (e.g. ϵG, ϵT) could likely be accommodated in the Tdp2 active site via planar base stacking with the active site facing hydrophobic wall of the β2Hβ motif.	RESULTS
181	197	hydrophobic wall	site	Therefore, structurally diverse undamaged or alkylated bases (e.g. ϵG, ϵT) could likely be accommodated in the Tdp2 active site via planar base stacking with the active site facing hydrophobic wall of the β2Hβ motif.	RESULTS
205	209	β2Hβ	structure_element	Therefore, structurally diverse undamaged or alkylated bases (e.g. ϵG, ϵT) could likely be accommodated in the Tdp2 active site via planar base stacking with the active site facing hydrophobic wall of the β2Hβ motif.	RESULTS
14	32	abasic deoxyribose	chemical	Likewise, the abasic deoxyribose analog THF substrate binds similar to the alkylated and non-alkylated substrates, but with a slight alteration in the approach of the 5′-terminus (Figure 2C).	RESULTS
40	43	THF	chemical	Likewise, the abasic deoxyribose analog THF substrate binds similar to the alkylated and non-alkylated substrates, but with a slight alteration in the approach of the 5′-terminus (Figure 2C).	RESULTS
22	32	absence of	protein_state	Interestingly, in the absence of a nucleobase, O4′ of the THF ring adopts a close approach (2.8 Å) to a water molecule that directly participates in the outer sphere single Mg2+ ion coordination shell (Figure 2D).	RESULTS
58	61	THF	chemical	Interestingly, in the absence of a nucleobase, O4′ of the THF ring adopts a close approach (2.8 Å) to a water molecule that directly participates in the outer sphere single Mg2+ ion coordination shell (Figure 2D).	RESULTS
104	109	water	chemical	Interestingly, in the absence of a nucleobase, O4′ of the THF ring adopts a close approach (2.8 Å) to a water molecule that directly participates in the outer sphere single Mg2+ ion coordination shell (Figure 2D).	RESULTS
173	177	Mg2+	chemical	Interestingly, in the absence of a nucleobase, O4′ of the THF ring adopts a close approach (2.8 Å) to a water molecule that directly participates in the outer sphere single Mg2+ ion coordination shell (Figure 2D).	RESULTS
178	200	ion coordination shell	bond_interaction	Interestingly, in the absence of a nucleobase, O4′ of the THF ring adopts a close approach (2.8 Å) to a water molecule that directly participates in the outer sphere single Mg2+ ion coordination shell (Figure 2D).	RESULTS
108	111	THF	chemical	These collective differences may explain the slight, but statistically significant elevated activity on the THF substrate (Figure 1C).	RESULTS
29	33	Tdp2	protein	Structural plasticity in the Tdp2 DNA binding trench	RESULTS
34	52	DNA binding trench	site	Structural plasticity in the Tdp2 DNA binding trench	RESULTS
29	45	DNA-damage bound	protein_state	An intriguing feature of the DNA-damage bound conformation of the Tdp2 active site is an underlying network of protein–water–protein contacts that span a gap between the catalytic core and the DNA binding β2Hβ-grasp (Supplementary Figure S2).	RESULTS
66	70	Tdp2	protein	An intriguing feature of the DNA-damage bound conformation of the Tdp2 active site is an underlying network of protein–water–protein contacts that span a gap between the catalytic core and the DNA binding β2Hβ-grasp (Supplementary Figure S2).	RESULTS
71	82	active site	site	An intriguing feature of the DNA-damage bound conformation of the Tdp2 active site is an underlying network of protein–water–protein contacts that span a gap between the catalytic core and the DNA binding β2Hβ-grasp (Supplementary Figure S2).	RESULTS
119	124	water	chemical	An intriguing feature of the DNA-damage bound conformation of the Tdp2 active site is an underlying network of protein–water–protein contacts that span a gap between the catalytic core and the DNA binding β2Hβ-grasp (Supplementary Figure S2).	RESULTS
170	184	catalytic core	site	An intriguing feature of the DNA-damage bound conformation of the Tdp2 active site is an underlying network of protein–water–protein contacts that span a gap between the catalytic core and the DNA binding β2Hβ-grasp (Supplementary Figure S2).	RESULTS
193	215	DNA binding β2Hβ-grasp	site	An intriguing feature of the DNA-damage bound conformation of the Tdp2 active site is an underlying network of protein–water–protein contacts that span a gap between the catalytic core and the DNA binding β2Hβ-grasp (Supplementary Figure S2).	RESULTS
68	78	β2Hβ-grasp	site	In this arrangement, six solvent molecules form a channel under the β2Hβ-grasp, ending with hydrogen bonds to the peptide backbone of the Mg2+ ligand Asp358.	RESULTS
92	106	hydrogen bonds	bond_interaction	In this arrangement, six solvent molecules form a channel under the β2Hβ-grasp, ending with hydrogen bonds to the peptide backbone of the Mg2+ ligand Asp358.	RESULTS
138	142	Mg2+	chemical	In this arrangement, six solvent molecules form a channel under the β2Hβ-grasp, ending with hydrogen bonds to the peptide backbone of the Mg2+ ligand Asp358.	RESULTS
150	156	Asp358	residue_name_number	In this arrangement, six solvent molecules form a channel under the β2Hβ-grasp, ending with hydrogen bonds to the peptide backbone of the Mg2+ ligand Asp358.	RESULTS
15	39	hydrophobic interactions	bond_interaction	The paucity of hydrophobic interactions stabilizing the β2Hβ DNA-bound conformation suggests that conformational plasticity in the β2Hβ might be a feature of DNA damage and metal cofactor engagement.	RESULTS
56	60	β2Hβ	structure_element	The paucity of hydrophobic interactions stabilizing the β2Hβ DNA-bound conformation suggests that conformational plasticity in the β2Hβ might be a feature of DNA damage and metal cofactor engagement.	RESULTS
61	70	DNA-bound	protein_state	The paucity of hydrophobic interactions stabilizing the β2Hβ DNA-bound conformation suggests that conformational plasticity in the β2Hβ might be a feature of DNA damage and metal cofactor engagement.	RESULTS
131	135	β2Hβ	structure_element	The paucity of hydrophobic interactions stabilizing the β2Hβ DNA-bound conformation suggests that conformational plasticity in the β2Hβ might be a feature of DNA damage and metal cofactor engagement.	RESULTS
158	161	DNA	chemical	The paucity of hydrophobic interactions stabilizing the β2Hβ DNA-bound conformation suggests that conformational plasticity in the β2Hβ might be a feature of DNA damage and metal cofactor engagement.	RESULTS
28	40	crystallized	experimental_method	To test this hypothesis, we crystallized Tdp2 in the absence of DNA and determined a DNA free Tdp2 structure to 2.4 Å resolution (PDB entry 5INM; Figures 1F and 3A).	RESULTS
41	45	Tdp2	protein	To test this hypothesis, we crystallized Tdp2 in the absence of DNA and determined a DNA free Tdp2 structure to 2.4 Å resolution (PDB entry 5INM; Figures 1F and 3A).	RESULTS
53	63	absence of	protein_state	To test this hypothesis, we crystallized Tdp2 in the absence of DNA and determined a DNA free Tdp2 structure to 2.4 Å resolution (PDB entry 5INM; Figures 1F and 3A).	RESULTS
64	67	DNA	chemical	To test this hypothesis, we crystallized Tdp2 in the absence of DNA and determined a DNA free Tdp2 structure to 2.4 Å resolution (PDB entry 5INM; Figures 1F and 3A).	RESULTS
85	93	DNA free	protein_state	To test this hypothesis, we crystallized Tdp2 in the absence of DNA and determined a DNA free Tdp2 structure to 2.4 Å resolution (PDB entry 5INM; Figures 1F and 3A).	RESULTS
94	98	Tdp2	protein	To test this hypothesis, we crystallized Tdp2 in the absence of DNA and determined a DNA free Tdp2 structure to 2.4 Å resolution (PDB entry 5INM; Figures 1F and 3A).	RESULTS
99	108	structure	evidence	To test this hypothesis, we crystallized Tdp2 in the absence of DNA and determined a DNA free Tdp2 structure to 2.4 Å resolution (PDB entry 5INM; Figures 1F and 3A).	RESULTS
33	37	Tdp2	protein	Conformational plasticity in the Tdp2 active site.	FIG
38	49	active site	site	Conformational plasticity in the Tdp2 active site.	FIG
8	12	open	protein_state	(A) The open, 3-helix conformation (tan) of flexible active-site loop observed in monomer E of the DNA-free mTdp2cat structure (PDB entry 5INM) is supported by T309 (green), which packs against the EEP core.	FIG
14	21	3-helix	structure_element	(A) The open, 3-helix conformation (tan) of flexible active-site loop observed in monomer E of the DNA-free mTdp2cat structure (PDB entry 5INM) is supported by T309 (green), which packs against the EEP core.	FIG
44	52	flexible	protein_state	(A) The open, 3-helix conformation (tan) of flexible active-site loop observed in monomer E of the DNA-free mTdp2cat structure (PDB entry 5INM) is supported by T309 (green), which packs against the EEP core.	FIG
53	69	active-site loop	structure_element	(A) The open, 3-helix conformation (tan) of flexible active-site loop observed in monomer E of the DNA-free mTdp2cat structure (PDB entry 5INM) is supported by T309 (green), which packs against the EEP core.	FIG
82	89	monomer	oligomeric_state	(A) The open, 3-helix conformation (tan) of flexible active-site loop observed in monomer E of the DNA-free mTdp2cat structure (PDB entry 5INM) is supported by T309 (green), which packs against the EEP core.	FIG
90	91	E	structure_element	(A) The open, 3-helix conformation (tan) of flexible active-site loop observed in monomer E of the DNA-free mTdp2cat structure (PDB entry 5INM) is supported by T309 (green), which packs against the EEP core.	FIG
99	107	DNA-free	protein_state	(A) The open, 3-helix conformation (tan) of flexible active-site loop observed in monomer E of the DNA-free mTdp2cat structure (PDB entry 5INM) is supported by T309 (green), which packs against the EEP core.	FIG
108	116	mTdp2cat	structure_element	(A) The open, 3-helix conformation (tan) of flexible active-site loop observed in monomer E of the DNA-free mTdp2cat structure (PDB entry 5INM) is supported by T309 (green), which packs against the EEP core.	FIG
117	126	structure	evidence	(A) The open, 3-helix conformation (tan) of flexible active-site loop observed in monomer E of the DNA-free mTdp2cat structure (PDB entry 5INM) is supported by T309 (green), which packs against the EEP core.	FIG
160	164	T309	residue_name_number	(A) The open, 3-helix conformation (tan) of flexible active-site loop observed in monomer E of the DNA-free mTdp2cat structure (PDB entry 5INM) is supported by T309 (green), which packs against the EEP core.	FIG
198	201	EEP	structure_element	(A) The open, 3-helix conformation (tan) of flexible active-site loop observed in monomer E of the DNA-free mTdp2cat structure (PDB entry 5INM) is supported by T309 (green), which packs against the EEP core.	FIG
4	23	β2Hβ docking pocket	site	The β2Hβ docking pocket (circled) is unoccupied and residues N312, N314 and L315 (orange) are solvent-exposed.	FIG
61	65	N312	residue_name_number	The β2Hβ docking pocket (circled) is unoccupied and residues N312, N314 and L315 (orange) are solvent-exposed.	FIG
67	71	N314	residue_name_number	The β2Hβ docking pocket (circled) is unoccupied and residues N312, N314 and L315 (orange) are solvent-exposed.	FIG
76	80	L315	residue_name_number	The β2Hβ docking pocket (circled) is unoccupied and residues N312, N314 and L315 (orange) are solvent-exposed.	FIG
94	109	solvent-exposed	protein_state	The β2Hβ docking pocket (circled) is unoccupied and residues N312, N314 and L315 (orange) are solvent-exposed.	FIG
44	50	closed	protein_state	Wall-eyed stereo view is displayed. (B) The closed β2Hβ conformation in the mTdp2cat–DNA product structure containing 5′-ϵA (yellow, PDB entry 5HT2). T309 (green) is an integral part of the β2Hβ DNA-binding grasp (tan) and hydrogen bonds to the backbone of Y321, while N314 (orange) occupies the β2Hβ docking pocket.	FIG
51	55	β2Hβ	structure_element	Wall-eyed stereo view is displayed. (B) The closed β2Hβ conformation in the mTdp2cat–DNA product structure containing 5′-ϵA (yellow, PDB entry 5HT2). T309 (green) is an integral part of the β2Hβ DNA-binding grasp (tan) and hydrogen bonds to the backbone of Y321, while N314 (orange) occupies the β2Hβ docking pocket.	FIG
76	88	mTdp2cat–DNA	complex_assembly	Wall-eyed stereo view is displayed. (B) The closed β2Hβ conformation in the mTdp2cat–DNA product structure containing 5′-ϵA (yellow, PDB entry 5HT2). T309 (green) is an integral part of the β2Hβ DNA-binding grasp (tan) and hydrogen bonds to the backbone of Y321, while N314 (orange) occupies the β2Hβ docking pocket.	FIG
97	106	structure	evidence	Wall-eyed stereo view is displayed. (B) The closed β2Hβ conformation in the mTdp2cat–DNA product structure containing 5′-ϵA (yellow, PDB entry 5HT2). T309 (green) is an integral part of the β2Hβ DNA-binding grasp (tan) and hydrogen bonds to the backbone of Y321, while N314 (orange) occupies the β2Hβ docking pocket.	FIG
118	123	5′-ϵA	chemical	Wall-eyed stereo view is displayed. (B) The closed β2Hβ conformation in the mTdp2cat–DNA product structure containing 5′-ϵA (yellow, PDB entry 5HT2). T309 (green) is an integral part of the β2Hβ DNA-binding grasp (tan) and hydrogen bonds to the backbone of Y321, while N314 (orange) occupies the β2Hβ docking pocket.	FIG
150	154	T309	residue_name_number	Wall-eyed stereo view is displayed. (B) The closed β2Hβ conformation in the mTdp2cat–DNA product structure containing 5′-ϵA (yellow, PDB entry 5HT2). T309 (green) is an integral part of the β2Hβ DNA-binding grasp (tan) and hydrogen bonds to the backbone of Y321, while N314 (orange) occupies the β2Hβ docking pocket.	FIG
190	212	β2Hβ DNA-binding grasp	site	Wall-eyed stereo view is displayed. (B) The closed β2Hβ conformation in the mTdp2cat–DNA product structure containing 5′-ϵA (yellow, PDB entry 5HT2). T309 (green) is an integral part of the β2Hβ DNA-binding grasp (tan) and hydrogen bonds to the backbone of Y321, while N314 (orange) occupies the β2Hβ docking pocket.	FIG
223	237	hydrogen bonds	bond_interaction	Wall-eyed stereo view is displayed. (B) The closed β2Hβ conformation in the mTdp2cat–DNA product structure containing 5′-ϵA (yellow, PDB entry 5HT2). T309 (green) is an integral part of the β2Hβ DNA-binding grasp (tan) and hydrogen bonds to the backbone of Y321, while N314 (orange) occupies the β2Hβ docking pocket.	FIG
257	261	Y321	residue_name_number	Wall-eyed stereo view is displayed. (B) The closed β2Hβ conformation in the mTdp2cat–DNA product structure containing 5′-ϵA (yellow, PDB entry 5HT2). T309 (green) is an integral part of the β2Hβ DNA-binding grasp (tan) and hydrogen bonds to the backbone of Y321, while N314 (orange) occupies the β2Hβ docking pocket.	FIG
269	273	N314	residue_name_number	Wall-eyed stereo view is displayed. (B) The closed β2Hβ conformation in the mTdp2cat–DNA product structure containing 5′-ϵA (yellow, PDB entry 5HT2). T309 (green) is an integral part of the β2Hβ DNA-binding grasp (tan) and hydrogen bonds to the backbone of Y321, while N314 (orange) occupies the β2Hβ docking pocket.	FIG
296	315	β2Hβ docking pocket	site	Wall-eyed stereo view is displayed. (B) The closed β2Hβ conformation in the mTdp2cat–DNA product structure containing 5′-ϵA (yellow, PDB entry 5HT2). T309 (green) is an integral part of the β2Hβ DNA-binding grasp (tan) and hydrogen bonds to the backbone of Y321, while N314 (orange) occupies the β2Hβ docking pocket.	FIG
53	69	active site loop	structure_element	Wall-eyed stereo view is displayed. (C) Alignment of active site loop conformers observed in the 5 promoters of the DNA-free mTdp2cat (PDB entry 5INM, see Table 1) crystallographic asymmetric unit (left) and sequence alignment showing residues not observed in the electron density as ‘∼’ (right). (D) Limited trypsin proteolysis probes the solvent accessibility of the flexible active-site loop.	FIG
99	108	promoters	oligomeric_state	Wall-eyed stereo view is displayed. (C) Alignment of active site loop conformers observed in the 5 promoters of the DNA-free mTdp2cat (PDB entry 5INM, see Table 1) crystallographic asymmetric unit (left) and sequence alignment showing residues not observed in the electron density as ‘∼’ (right). (D) Limited trypsin proteolysis probes the solvent accessibility of the flexible active-site loop.	FIG
116	124	DNA-free	protein_state	Wall-eyed stereo view is displayed. (C) Alignment of active site loop conformers observed in the 5 promoters of the DNA-free mTdp2cat (PDB entry 5INM, see Table 1) crystallographic asymmetric unit (left) and sequence alignment showing residues not observed in the electron density as ‘∼’ (right). (D) Limited trypsin proteolysis probes the solvent accessibility of the flexible active-site loop.	FIG
125	133	mTdp2cat	structure_element	Wall-eyed stereo view is displayed. (C) Alignment of active site loop conformers observed in the 5 promoters of the DNA-free mTdp2cat (PDB entry 5INM, see Table 1) crystallographic asymmetric unit (left) and sequence alignment showing residues not observed in the electron density as ‘∼’ (right). (D) Limited trypsin proteolysis probes the solvent accessibility of the flexible active-site loop.	FIG
208	226	sequence alignment	experimental_method	Wall-eyed stereo view is displayed. (C) Alignment of active site loop conformers observed in the 5 promoters of the DNA-free mTdp2cat (PDB entry 5INM, see Table 1) crystallographic asymmetric unit (left) and sequence alignment showing residues not observed in the electron density as ‘∼’ (right). (D) Limited trypsin proteolysis probes the solvent accessibility of the flexible active-site loop.	FIG
264	280	electron density	evidence	Wall-eyed stereo view is displayed. (C) Alignment of active site loop conformers observed in the 5 promoters of the DNA-free mTdp2cat (PDB entry 5INM, see Table 1) crystallographic asymmetric unit (left) and sequence alignment showing residues not observed in the electron density as ‘∼’ (right). (D) Limited trypsin proteolysis probes the solvent accessibility of the flexible active-site loop.	FIG
301	328	Limited trypsin proteolysis	experimental_method	Wall-eyed stereo view is displayed. (C) Alignment of active site loop conformers observed in the 5 promoters of the DNA-free mTdp2cat (PDB entry 5INM, see Table 1) crystallographic asymmetric unit (left) and sequence alignment showing residues not observed in the electron density as ‘∼’ (right). (D) Limited trypsin proteolysis probes the solvent accessibility of the flexible active-site loop.	FIG
369	377	flexible	protein_state	Wall-eyed stereo view is displayed. (C) Alignment of active site loop conformers observed in the 5 promoters of the DNA-free mTdp2cat (PDB entry 5INM, see Table 1) crystallographic asymmetric unit (left) and sequence alignment showing residues not observed in the electron density as ‘∼’ (right). (D) Limited trypsin proteolysis probes the solvent accessibility of the flexible active-site loop.	FIG
378	394	active-site loop	structure_element	Wall-eyed stereo view is displayed. (C) Alignment of active site loop conformers observed in the 5 promoters of the DNA-free mTdp2cat (PDB entry 5INM, see Table 1) crystallographic asymmetric unit (left) and sequence alignment showing residues not observed in the electron density as ‘∼’ (right). (D) Limited trypsin proteolysis probes the solvent accessibility of the flexible active-site loop.	FIG
0	8	mTdp2cat	structure_element	mTdp2cat WT (lanes 1–13) or mTdp2cat D358N (lanes 14–26) were incubated in the presence or absence of Mg2+ and/or a 12 nt self annealing, 5′-phosphorylated DNA (substrate ‘12 nt’ in Supplementary Table S1), then reacted with 0.6, 1.7 or 5 ng μl−1 of trypsin.	FIG
9	11	WT	protein_state	mTdp2cat WT (lanes 1–13) or mTdp2cat D358N (lanes 14–26) were incubated in the presence or absence of Mg2+ and/or a 12 nt self annealing, 5′-phosphorylated DNA (substrate ‘12 nt’ in Supplementary Table S1), then reacted with 0.6, 1.7 or 5 ng μl−1 of trypsin.	FIG
28	36	mTdp2cat	structure_element	mTdp2cat WT (lanes 1–13) or mTdp2cat D358N (lanes 14–26) were incubated in the presence or absence of Mg2+ and/or a 12 nt self annealing, 5′-phosphorylated DNA (substrate ‘12 nt’ in Supplementary Table S1), then reacted with 0.6, 1.7 or 5 ng μl−1 of trypsin.	FIG
37	42	D358N	mutant	mTdp2cat WT (lanes 1–13) or mTdp2cat D358N (lanes 14–26) were incubated in the presence or absence of Mg2+ and/or a 12 nt self annealing, 5′-phosphorylated DNA (substrate ‘12 nt’ in Supplementary Table S1), then reacted with 0.6, 1.7 or 5 ng μl−1 of trypsin.	FIG
79	87	presence	protein_state	mTdp2cat WT (lanes 1–13) or mTdp2cat D358N (lanes 14–26) were incubated in the presence or absence of Mg2+ and/or a 12 nt self annealing, 5′-phosphorylated DNA (substrate ‘12 nt’ in Supplementary Table S1), then reacted with 0.6, 1.7 or 5 ng μl−1 of trypsin.	FIG
91	101	absence of	protein_state	mTdp2cat WT (lanes 1–13) or mTdp2cat D358N (lanes 14–26) were incubated in the presence or absence of Mg2+ and/or a 12 nt self annealing, 5′-phosphorylated DNA (substrate ‘12 nt’ in Supplementary Table S1), then reacted with 0.6, 1.7 or 5 ng μl−1 of trypsin.	FIG
102	106	Mg2+	chemical	mTdp2cat WT (lanes 1–13) or mTdp2cat D358N (lanes 14–26) were incubated in the presence or absence of Mg2+ and/or a 12 nt self annealing, 5′-phosphorylated DNA (substrate ‘12 nt’ in Supplementary Table S1), then reacted with 0.6, 1.7 or 5 ng μl−1 of trypsin.	FIG
156	159	DNA	chemical	mTdp2cat WT (lanes 1–13) or mTdp2cat D358N (lanes 14–26) were incubated in the presence or absence of Mg2+ and/or a 12 nt self annealing, 5′-phosphorylated DNA (substrate ‘12 nt’ in Supplementary Table S1), then reacted with 0.6, 1.7 or 5 ng μl−1 of trypsin.	FIG
28	36	SDS-PAGE	experimental_method	Reactions were separated by SDS-PAGE and proteins visualized by staining with coomassie blue. (E) Limited chymotrypsin proteolysis probes the solvent accessibility of the flexible active-site loop.	FIG
98	130	Limited chymotrypsin proteolysis	experimental_method	Reactions were separated by SDS-PAGE and proteins visualized by staining with coomassie blue. (E) Limited chymotrypsin proteolysis probes the solvent accessibility of the flexible active-site loop.	FIG
171	179	flexible	protein_state	Reactions were separated by SDS-PAGE and proteins visualized by staining with coomassie blue. (E) Limited chymotrypsin proteolysis probes the solvent accessibility of the flexible active-site loop.	FIG
180	196	active-site loop	structure_element	Reactions were separated by SDS-PAGE and proteins visualized by staining with coomassie blue. (E) Limited chymotrypsin proteolysis probes the solvent accessibility of the flexible active-site loop.	FIG
40	48	mTdp2cat	structure_element	Experiments performed as in panel D for mTdp2cat WT (lanes 27–39) or mTdp2cat D358N (lanes 40–52), but with chymotrypsin instead of trypsin.	FIG
49	51	WT	protein_state	Experiments performed as in panel D for mTdp2cat WT (lanes 27–39) or mTdp2cat D358N (lanes 40–52), but with chymotrypsin instead of trypsin.	FIG
69	77	mTdp2cat	structure_element	Experiments performed as in panel D for mTdp2cat WT (lanes 27–39) or mTdp2cat D358N (lanes 40–52), but with chymotrypsin instead of trypsin.	FIG
78	83	D358N	mutant	Experiments performed as in panel D for mTdp2cat WT (lanes 27–39) or mTdp2cat D358N (lanes 40–52), but with chymotrypsin instead of trypsin.	FIG
108	120	chymotrypsin	experimental_method	Experiments performed as in panel D for mTdp2cat WT (lanes 27–39) or mTdp2cat D358N (lanes 40–52), but with chymotrypsin instead of trypsin.	FIG
132	139	trypsin	experimental_method	Experiments performed as in panel D for mTdp2cat WT (lanes 27–39) or mTdp2cat D358N (lanes 40–52), but with chymotrypsin instead of trypsin.	FIG
29	33	Tdp2	protein	This crystal form contains 5 Tdp2 protein molecules in the asymmetric unit, with variations in active site Mg2+ occupancy and substrate binding loops observed for the individual protomers.	RESULTS
95	106	active site	site	This crystal form contains 5 Tdp2 protein molecules in the asymmetric unit, with variations in active site Mg2+ occupancy and substrate binding loops observed for the individual protomers.	RESULTS
107	111	Mg2+	chemical	This crystal form contains 5 Tdp2 protein molecules in the asymmetric unit, with variations in active site Mg2+ occupancy and substrate binding loops observed for the individual protomers.	RESULTS
126	149	substrate binding loops	structure_element	This crystal form contains 5 Tdp2 protein molecules in the asymmetric unit, with variations in active site Mg2+ occupancy and substrate binding loops observed for the individual protomers.	RESULTS
178	187	protomers	oligomeric_state	This crystal form contains 5 Tdp2 protein molecules in the asymmetric unit, with variations in active site Mg2+ occupancy and substrate binding loops observed for the individual protomers.	RESULTS
33	48	DNA ligand-free	protein_state	The most striking feature of the DNA ligand-free state is that the active site β2Hβ-grasp can adopt alternative structures that are distinct from the DNA-bound, closed β2Hβ DNA binding grasp (Figure 3A and B).	RESULTS
67	89	active site β2Hβ-grasp	site	The most striking feature of the DNA ligand-free state is that the active site β2Hβ-grasp can adopt alternative structures that are distinct from the DNA-bound, closed β2Hβ DNA binding grasp (Figure 3A and B).	RESULTS
150	159	DNA-bound	protein_state	The most striking feature of the DNA ligand-free state is that the active site β2Hβ-grasp can adopt alternative structures that are distinct from the DNA-bound, closed β2Hβ DNA binding grasp (Figure 3A and B).	RESULTS
161	167	closed	protein_state	The most striking feature of the DNA ligand-free state is that the active site β2Hβ-grasp can adopt alternative structures that are distinct from the DNA-bound, closed β2Hβ DNA binding grasp (Figure 3A and B).	RESULTS
168	190	β2Hβ DNA binding grasp	site	The most striking feature of the DNA ligand-free state is that the active site β2Hβ-grasp can adopt alternative structures that are distinct from the DNA-bound, closed β2Hβ DNA binding grasp (Figure 3A and B).	RESULTS
7	14	monomer	oligomeric_state	In one monomer (chain ‘E’), the grasp adopts an ‘open’ 3-helix loop conformation that projects away from the EEP catalytic core.	RESULTS
16	25	chain ‘E’	structure_element	In one monomer (chain ‘E’), the grasp adopts an ‘open’ 3-helix loop conformation that projects away from the EEP catalytic core.	RESULTS
32	37	grasp	structure_element	In one monomer (chain ‘E’), the grasp adopts an ‘open’ 3-helix loop conformation that projects away from the EEP catalytic core.	RESULTS
49	53	open	protein_state	In one monomer (chain ‘E’), the grasp adopts an ‘open’ 3-helix loop conformation that projects away from the EEP catalytic core.	RESULTS
55	67	3-helix loop	structure_element	In one monomer (chain ‘E’), the grasp adopts an ‘open’ 3-helix loop conformation that projects away from the EEP catalytic core.	RESULTS
109	112	EEP	structure_element	In one monomer (chain ‘E’), the grasp adopts an ‘open’ 3-helix loop conformation that projects away from the EEP catalytic core.	RESULTS
113	127	catalytic core	site	In one monomer (chain ‘E’), the grasp adopts an ‘open’ 3-helix loop conformation that projects away from the EEP catalytic core.	RESULTS
4	12	monomers	oligomeric_state	Two monomers have variable disordered states for which much of the DNA binding loop is not visible in the electron density.	RESULTS
27	37	disordered	protein_state	Two monomers have variable disordered states for which much of the DNA binding loop is not visible in the electron density.	RESULTS
67	83	DNA binding loop	structure_element	Two monomers have variable disordered states for which much of the DNA binding loop is not visible in the electron density.	RESULTS
106	122	electron density	evidence	Two monomers have variable disordered states for which much of the DNA binding loop is not visible in the electron density.	RESULTS
35	43	DNA-free	protein_state	The remaining two molecules in the DNA-free crystal form are closed β2Hβ conformers similar to the DNA bound structures (Figure 3C).	RESULTS
44	56	crystal form	evidence	The remaining two molecules in the DNA-free crystal form are closed β2Hβ conformers similar to the DNA bound structures (Figure 3C).	RESULTS
61	67	closed	protein_state	The remaining two molecules in the DNA-free crystal form are closed β2Hβ conformers similar to the DNA bound structures (Figure 3C).	RESULTS
68	72	β2Hβ	structure_element	The remaining two molecules in the DNA-free crystal form are closed β2Hβ conformers similar to the DNA bound structures (Figure 3C).	RESULTS
99	108	DNA bound	protein_state	The remaining two molecules in the DNA-free crystal form are closed β2Hβ conformers similar to the DNA bound structures (Figure 3C).	RESULTS
109	119	structures	evidence	The remaining two molecules in the DNA-free crystal form are closed β2Hβ conformers similar to the DNA bound structures (Figure 3C).	RESULTS
20	24	Tdp2	protein	Thus, we posit that Tdp2 DNA binding conformationally selects the closed form of the β2Hβ grasp, rather than inducing closure upon binding.	RESULTS
25	28	DNA	chemical	Thus, we posit that Tdp2 DNA binding conformationally selects the closed form of the β2Hβ grasp, rather than inducing closure upon binding.	RESULTS
66	72	closed	protein_state	Thus, we posit that Tdp2 DNA binding conformationally selects the closed form of the β2Hβ grasp, rather than inducing closure upon binding.	RESULTS
85	95	β2Hβ grasp	site	Thus, we posit that Tdp2 DNA binding conformationally selects the closed form of the β2Hβ grasp, rather than inducing closure upon binding.	RESULTS
27	35	extended	protein_state	A detailed analysis of the extended 3-helix conformation shows that the substrate-binding loop is able to undergo metamorphic structural changes.	RESULTS
36	43	3-helix	structure_element	A detailed analysis of the extended 3-helix conformation shows that the substrate-binding loop is able to undergo metamorphic structural changes.	RESULTS
72	94	substrate-binding loop	structure_element	A detailed analysis of the extended 3-helix conformation shows that the substrate-binding loop is able to undergo metamorphic structural changes.	RESULTS
8	12	open	protein_state	In this open form, residues Asn312-Leu315 are distal from the active site and solvent-exposed (orange sticks, Figure 3A), while Thr309 (green surface, Figure 3A) packs into a shallow pocket of the EEP core to anchor the loop.	RESULTS
28	41	Asn312-Leu315	residue_range	In this open form, residues Asn312-Leu315 are distal from the active site and solvent-exposed (orange sticks, Figure 3A), while Thr309 (green surface, Figure 3A) packs into a shallow pocket of the EEP core to anchor the loop.	RESULTS
62	73	active site	site	In this open form, residues Asn312-Leu315 are distal from the active site and solvent-exposed (orange sticks, Figure 3A), while Thr309 (green surface, Figure 3A) packs into a shallow pocket of the EEP core to anchor the loop.	RESULTS
78	93	solvent-exposed	protein_state	In this open form, residues Asn312-Leu315 are distal from the active site and solvent-exposed (orange sticks, Figure 3A), while Thr309 (green surface, Figure 3A) packs into a shallow pocket of the EEP core to anchor the loop.	RESULTS
128	134	Thr309	residue_name_number	In this open form, residues Asn312-Leu315 are distal from the active site and solvent-exposed (orange sticks, Figure 3A), while Thr309 (green surface, Figure 3A) packs into a shallow pocket of the EEP core to anchor the loop.	RESULTS
183	189	pocket	site	In this open form, residues Asn312-Leu315 are distal from the active site and solvent-exposed (orange sticks, Figure 3A), while Thr309 (green surface, Figure 3A) packs into a shallow pocket of the EEP core to anchor the loop.	RESULTS
197	200	EEP	structure_element	In this open form, residues Asn312-Leu315 are distal from the active site and solvent-exposed (orange sticks, Figure 3A), while Thr309 (green surface, Figure 3A) packs into a shallow pocket of the EEP core to anchor the loop.	RESULTS
220	224	loop	structure_element	In this open form, residues Asn312-Leu315 are distal from the active site and solvent-exposed (orange sticks, Figure 3A), while Thr309 (green surface, Figure 3A) packs into a shallow pocket of the EEP core to anchor the loop.	RESULTS
10	16	Thr309	residue_name_number	Burial of Thr309 is enabled by an unusual main chain cis–peptide bond between Asp308-Thr309 and disassembly of the short antiparallel beta-strand of the β2Hβ fold.	RESULTS
53	69	cis–peptide bond	bond_interaction	Burial of Thr309 is enabled by an unusual main chain cis–peptide bond between Asp308-Thr309 and disassembly of the short antiparallel beta-strand of the β2Hβ fold.	RESULTS
78	84	Asp308	residue_name_number	Burial of Thr309 is enabled by an unusual main chain cis–peptide bond between Asp308-Thr309 and disassembly of the short antiparallel beta-strand of the β2Hβ fold.	RESULTS
85	91	Thr309	residue_name_number	Burial of Thr309 is enabled by an unusual main chain cis–peptide bond between Asp308-Thr309 and disassembly of the short antiparallel beta-strand of the β2Hβ fold.	RESULTS
115	145	short antiparallel beta-strand	structure_element	Burial of Thr309 is enabled by an unusual main chain cis–peptide bond between Asp308-Thr309 and disassembly of the short antiparallel beta-strand of the β2Hβ fold.	RESULTS
153	157	β2Hβ	structure_element	Burial of Thr309 is enabled by an unusual main chain cis–peptide bond between Asp308-Thr309 and disassembly of the short antiparallel beta-strand of the β2Hβ fold.	RESULTS
19	25	closed	protein_state	By comparison, the closed β2Hβ grasp conformer is stabilized by Asn312 and Asn314 binding into two β2Hβ docking pockets, and Leu315 engagement of the 5′-terminal nucleobase (Figure 3B).	RESULTS
26	36	β2Hβ grasp	site	By comparison, the closed β2Hβ grasp conformer is stabilized by Asn312 and Asn314 binding into two β2Hβ docking pockets, and Leu315 engagement of the 5′-terminal nucleobase (Figure 3B).	RESULTS
64	70	Asn312	residue_name_number	By comparison, the closed β2Hβ grasp conformer is stabilized by Asn312 and Asn314 binding into two β2Hβ docking pockets, and Leu315 engagement of the 5′-terminal nucleobase (Figure 3B).	RESULTS
75	81	Asn314	residue_name_number	By comparison, the closed β2Hβ grasp conformer is stabilized by Asn312 and Asn314 binding into two β2Hβ docking pockets, and Leu315 engagement of the 5′-terminal nucleobase (Figure 3B).	RESULTS
99	119	β2Hβ docking pockets	site	By comparison, the closed β2Hβ grasp conformer is stabilized by Asn312 and Asn314 binding into two β2Hβ docking pockets, and Leu315 engagement of the 5′-terminal nucleobase (Figure 3B).	RESULTS
125	131	Leu315	residue_name_number	By comparison, the closed β2Hβ grasp conformer is stabilized by Asn312 and Asn314 binding into two β2Hβ docking pockets, and Leu315 engagement of the 5′-terminal nucleobase (Figure 3B).	RESULTS
23	29	closed	protein_state	To transition into the closed β2Hβ conformation, Thr309 disengages from the EEP domain pocket, flips peptide backbone conformation cis to trans, and is integral to the β2Hβ antiparallel β-sheet.	RESULTS
30	34	β2Hβ	structure_element	To transition into the closed β2Hβ conformation, Thr309 disengages from the EEP domain pocket, flips peptide backbone conformation cis to trans, and is integral to the β2Hβ antiparallel β-sheet.	RESULTS
49	55	Thr309	residue_name_number	To transition into the closed β2Hβ conformation, Thr309 disengages from the EEP domain pocket, flips peptide backbone conformation cis to trans, and is integral to the β2Hβ antiparallel β-sheet.	RESULTS
76	79	EEP	structure_element	To transition into the closed β2Hβ conformation, Thr309 disengages from the EEP domain pocket, flips peptide backbone conformation cis to trans, and is integral to the β2Hβ antiparallel β-sheet.	RESULTS
87	93	pocket	site	To transition into the closed β2Hβ conformation, Thr309 disengages from the EEP domain pocket, flips peptide backbone conformation cis to trans, and is integral to the β2Hβ antiparallel β-sheet.	RESULTS
168	172	β2Hβ	structure_element	To transition into the closed β2Hβ conformation, Thr309 disengages from the EEP domain pocket, flips peptide backbone conformation cis to trans, and is integral to the β2Hβ antiparallel β-sheet.	RESULTS
173	193	antiparallel β-sheet	structure_element	To transition into the closed β2Hβ conformation, Thr309 disengages from the EEP domain pocket, flips peptide backbone conformation cis to trans, and is integral to the β2Hβ antiparallel β-sheet.	RESULTS
21	27	closed	protein_state	Stabilization of the closed β2Hβ-grasp conformation is linked to the active site through a hydrogen bond between Trp307 and the Mg2+ coordinating residue Asp358.	RESULTS
28	38	β2Hβ-grasp	site	Stabilization of the closed β2Hβ-grasp conformation is linked to the active site through a hydrogen bond between Trp307 and the Mg2+ coordinating residue Asp358.	RESULTS
69	80	active site	site	Stabilization of the closed β2Hβ-grasp conformation is linked to the active site through a hydrogen bond between Trp307 and the Mg2+ coordinating residue Asp358.	RESULTS
91	104	hydrogen bond	bond_interaction	Stabilization of the closed β2Hβ-grasp conformation is linked to the active site through a hydrogen bond between Trp307 and the Mg2+ coordinating residue Asp358.	RESULTS
113	119	Trp307	residue_name_number	Stabilization of the closed β2Hβ-grasp conformation is linked to the active site through a hydrogen bond between Trp307 and the Mg2+ coordinating residue Asp358.	RESULTS
128	153	Mg2+ coordinating residue	site	Stabilization of the closed β2Hβ-grasp conformation is linked to the active site through a hydrogen bond between Trp307 and the Mg2+ coordinating residue Asp358.	RESULTS
154	160	Asp358	residue_name_number	Stabilization of the closed β2Hβ-grasp conformation is linked to the active site through a hydrogen bond between Trp307 and the Mg2+ coordinating residue Asp358.	RESULTS
20	28	DNA free	protein_state	Accordingly, in the DNA free structure, we observe a trend where the 2 closed monomers have an ordered Mg2+ ion in their active sites, while the monomers with open conformations have a poorly ordered or vacant metal binding site.	RESULTS
29	38	structure	evidence	Accordingly, in the DNA free structure, we observe a trend where the 2 closed monomers have an ordered Mg2+ ion in their active sites, while the monomers with open conformations have a poorly ordered or vacant metal binding site.	RESULTS
71	77	closed	protein_state	Accordingly, in the DNA free structure, we observe a trend where the 2 closed monomers have an ordered Mg2+ ion in their active sites, while the monomers with open conformations have a poorly ordered or vacant metal binding site.	RESULTS
78	86	monomers	oligomeric_state	Accordingly, in the DNA free structure, we observe a trend where the 2 closed monomers have an ordered Mg2+ ion in their active sites, while the monomers with open conformations have a poorly ordered or vacant metal binding site.	RESULTS
103	107	Mg2+	chemical	Accordingly, in the DNA free structure, we observe a trend where the 2 closed monomers have an ordered Mg2+ ion in their active sites, while the monomers with open conformations have a poorly ordered or vacant metal binding site.	RESULTS
121	133	active sites	site	Accordingly, in the DNA free structure, we observe a trend where the 2 closed monomers have an ordered Mg2+ ion in their active sites, while the monomers with open conformations have a poorly ordered or vacant metal binding site.	RESULTS
145	153	monomers	oligomeric_state	Accordingly, in the DNA free structure, we observe a trend where the 2 closed monomers have an ordered Mg2+ ion in their active sites, while the monomers with open conformations have a poorly ordered or vacant metal binding site.	RESULTS
159	163	open	protein_state	Accordingly, in the DNA free structure, we observe a trend where the 2 closed monomers have an ordered Mg2+ ion in their active sites, while the monomers with open conformations have a poorly ordered or vacant metal binding site.	RESULTS
210	228	metal binding site	site	Accordingly, in the DNA free structure, we observe a trend where the 2 closed monomers have an ordered Mg2+ ion in their active sites, while the monomers with open conformations have a poorly ordered or vacant metal binding site.	RESULTS
71	74	DNA	chemical	Overall, these observations suggest that engagement of diverse damaged DNA ends is enabled by an elaborate substrate selected stabilization of the β2Hβ DNA binding grasp, and these rearrangements are coordinated with Mg2+ binding in the Tdp2 active site.	RESULTS
147	169	β2Hβ DNA binding grasp	site	Overall, these observations suggest that engagement of diverse damaged DNA ends is enabled by an elaborate substrate selected stabilization of the β2Hβ DNA binding grasp, and these rearrangements are coordinated with Mg2+ binding in the Tdp2 active site.	RESULTS
217	221	Mg2+	chemical	Overall, these observations suggest that engagement of diverse damaged DNA ends is enabled by an elaborate substrate selected stabilization of the β2Hβ DNA binding grasp, and these rearrangements are coordinated with Mg2+ binding in the Tdp2 active site.	RESULTS
237	241	Tdp2	protein	Overall, these observations suggest that engagement of diverse damaged DNA ends is enabled by an elaborate substrate selected stabilization of the β2Hβ DNA binding grasp, and these rearrangements are coordinated with Mg2+ binding in the Tdp2 active site.	RESULTS
242	253	active site	site	Overall, these observations suggest that engagement of diverse damaged DNA ends is enabled by an elaborate substrate selected stabilization of the β2Hβ DNA binding grasp, and these rearrangements are coordinated with Mg2+ binding in the Tdp2 active site.	RESULTS
12	16	Mg2+	chemical	To evaluate Mg2+ and DNA-dependent Tdp2 structural states in solution, we probed mTdp2cat conformations using limited trypsin and chymotrypsin proteolysis (Figure 3C–E).	RESULTS
21	24	DNA	chemical	To evaluate Mg2+ and DNA-dependent Tdp2 structural states in solution, we probed mTdp2cat conformations using limited trypsin and chymotrypsin proteolysis (Figure 3C–E).	RESULTS
35	39	Tdp2	protein	To evaluate Mg2+ and DNA-dependent Tdp2 structural states in solution, we probed mTdp2cat conformations using limited trypsin and chymotrypsin proteolysis (Figure 3C–E).	RESULTS
81	89	mTdp2cat	structure_element	To evaluate Mg2+ and DNA-dependent Tdp2 structural states in solution, we probed mTdp2cat conformations using limited trypsin and chymotrypsin proteolysis (Figure 3C–E).	RESULTS
110	154	limited trypsin and chymotrypsin proteolysis	experimental_method	To evaluate Mg2+ and DNA-dependent Tdp2 structural states in solution, we probed mTdp2cat conformations using limited trypsin and chymotrypsin proteolysis (Figure 3C–E).	RESULTS
0	17	In the absence of	protein_state	In the absence of DNA or Mg2+, mTdp2cat is efficiently cleaved in the metamorphic DNA binding grasp at one site by trypsin (Arg316), or at two positions by chymotrypsin (Trp307 and Leu315).	RESULTS
18	21	DNA	chemical	In the absence of DNA or Mg2+, mTdp2cat is efficiently cleaved in the metamorphic DNA binding grasp at one site by trypsin (Arg316), or at two positions by chymotrypsin (Trp307 and Leu315).	RESULTS
25	30	Mg2+,	chemical	In the absence of DNA or Mg2+, mTdp2cat is efficiently cleaved in the metamorphic DNA binding grasp at one site by trypsin (Arg316), or at two positions by chymotrypsin (Trp307 and Leu315).	RESULTS
31	39	mTdp2cat	structure_element	In the absence of DNA or Mg2+, mTdp2cat is efficiently cleaved in the metamorphic DNA binding grasp at one site by trypsin (Arg316), or at two positions by chymotrypsin (Trp307 and Leu315).	RESULTS
82	99	DNA binding grasp	site	In the absence of DNA or Mg2+, mTdp2cat is efficiently cleaved in the metamorphic DNA binding grasp at one site by trypsin (Arg316), or at two positions by chymotrypsin (Trp307 and Leu315).	RESULTS
115	122	trypsin	experimental_method	In the absence of DNA or Mg2+, mTdp2cat is efficiently cleaved in the metamorphic DNA binding grasp at one site by trypsin (Arg316), or at two positions by chymotrypsin (Trp307 and Leu315).	RESULTS
124	130	Arg316	residue_name_number	In the absence of DNA or Mg2+, mTdp2cat is efficiently cleaved in the metamorphic DNA binding grasp at one site by trypsin (Arg316), or at two positions by chymotrypsin (Trp307 and Leu315).	RESULTS
156	168	chymotrypsin	experimental_method	In the absence of DNA or Mg2+, mTdp2cat is efficiently cleaved in the metamorphic DNA binding grasp at one site by trypsin (Arg316), or at two positions by chymotrypsin (Trp307 and Leu315).	RESULTS
170	176	Trp307	residue_name_number	In the absence of DNA or Mg2+, mTdp2cat is efficiently cleaved in the metamorphic DNA binding grasp at one site by trypsin (Arg316), or at two positions by chymotrypsin (Trp307 and Leu315).	RESULTS
181	187	Leu315	residue_name_number	In the absence of DNA or Mg2+, mTdp2cat is efficiently cleaved in the metamorphic DNA binding grasp at one site by trypsin (Arg316), or at two positions by chymotrypsin (Trp307 and Leu315).	RESULTS
15	20	Mg2+,	chemical	By comparison, Mg2+, and to a greater extent Mg2+/DNA mixtures (compare Figure 3, lanes 4, 7 and 13) protect mTdp2cat from proteolytic cleavage.	RESULTS
45	49	Mg2+	chemical	By comparison, Mg2+, and to a greater extent Mg2+/DNA mixtures (compare Figure 3, lanes 4, 7 and 13) protect mTdp2cat from proteolytic cleavage.	RESULTS
50	53	DNA	chemical	By comparison, Mg2+, and to a greater extent Mg2+/DNA mixtures (compare Figure 3, lanes 4, 7 and 13) protect mTdp2cat from proteolytic cleavage.	RESULTS
109	117	mTdp2cat	structure_element	By comparison, Mg2+, and to a greater extent Mg2+/DNA mixtures (compare Figure 3, lanes 4, 7 and 13) protect mTdp2cat from proteolytic cleavage.	RESULTS
27	31	Mg2+	chemical	Interestingly, addition of Mg2+ alone protects against proteolysis as well.	RESULTS
24	28	Mg2+	chemical	This is consistent with Mg2+ stabilizing the closed conformation of the β2Hβ-grasp through an extended hydrogen-bonding network with Asp358 and the indole ring of the β2Hβ-grasp residue Trp307 (also discussion below on Tdp2 active site SNPs).	RESULTS
45	51	closed	protein_state	This is consistent with Mg2+ stabilizing the closed conformation of the β2Hβ-grasp through an extended hydrogen-bonding network with Asp358 and the indole ring of the β2Hβ-grasp residue Trp307 (also discussion below on Tdp2 active site SNPs).	RESULTS
72	82	β2Hβ-grasp	site	This is consistent with Mg2+ stabilizing the closed conformation of the β2Hβ-grasp through an extended hydrogen-bonding network with Asp358 and the indole ring of the β2Hβ-grasp residue Trp307 (also discussion below on Tdp2 active site SNPs).	RESULTS
103	127	hydrogen-bonding network	bond_interaction	This is consistent with Mg2+ stabilizing the closed conformation of the β2Hβ-grasp through an extended hydrogen-bonding network with Asp358 and the indole ring of the β2Hβ-grasp residue Trp307 (also discussion below on Tdp2 active site SNPs).	RESULTS
133	139	Asp358	residue_name_number	This is consistent with Mg2+ stabilizing the closed conformation of the β2Hβ-grasp through an extended hydrogen-bonding network with Asp358 and the indole ring of the β2Hβ-grasp residue Trp307 (also discussion below on Tdp2 active site SNPs).	RESULTS
167	177	β2Hβ-grasp	site	This is consistent with Mg2+ stabilizing the closed conformation of the β2Hβ-grasp through an extended hydrogen-bonding network with Asp358 and the indole ring of the β2Hβ-grasp residue Trp307 (also discussion below on Tdp2 active site SNPs).	RESULTS
186	192	Trp307	residue_name_number	This is consistent with Mg2+ stabilizing the closed conformation of the β2Hβ-grasp through an extended hydrogen-bonding network with Asp358 and the indole ring of the β2Hβ-grasp residue Trp307 (also discussion below on Tdp2 active site SNPs).	RESULTS
219	223	Tdp2	protein	This is consistent with Mg2+ stabilizing the closed conformation of the β2Hβ-grasp through an extended hydrogen-bonding network with Asp358 and the indole ring of the β2Hβ-grasp residue Trp307 (also discussion below on Tdp2 active site SNPs).	RESULTS
224	235	active site	site	This is consistent with Mg2+ stabilizing the closed conformation of the β2Hβ-grasp through an extended hydrogen-bonding network with Asp358 and the indole ring of the β2Hβ-grasp residue Trp307 (also discussion below on Tdp2 active site SNPs).	RESULTS
37	41	Tdp2	protein	To assess structural conservation of Tdp2 conformational changes between human and mouse Tdp2, we also determined a 3.2 Å resolution structure of the human Tdp2cat domain bound to a DNA 5′-PO4 terminus product complex (PDB entry 5INO).	RESULTS
73	78	human	species	To assess structural conservation of Tdp2 conformational changes between human and mouse Tdp2, we also determined a 3.2 Å resolution structure of the human Tdp2cat domain bound to a DNA 5′-PO4 terminus product complex (PDB entry 5INO).	RESULTS
83	88	mouse	taxonomy_domain	To assess structural conservation of Tdp2 conformational changes between human and mouse Tdp2, we also determined a 3.2 Å resolution structure of the human Tdp2cat domain bound to a DNA 5′-PO4 terminus product complex (PDB entry 5INO).	RESULTS
89	93	Tdp2	protein	To assess structural conservation of Tdp2 conformational changes between human and mouse Tdp2, we also determined a 3.2 Å resolution structure of the human Tdp2cat domain bound to a DNA 5′-PO4 terminus product complex (PDB entry 5INO).	RESULTS
103	113	determined	experimental_method	To assess structural conservation of Tdp2 conformational changes between human and mouse Tdp2, we also determined a 3.2 Å resolution structure of the human Tdp2cat domain bound to a DNA 5′-PO4 terminus product complex (PDB entry 5INO).	RESULTS
133	142	structure	evidence	To assess structural conservation of Tdp2 conformational changes between human and mouse Tdp2, we also determined a 3.2 Å resolution structure of the human Tdp2cat domain bound to a DNA 5′-PO4 terminus product complex (PDB entry 5INO).	RESULTS
150	155	human	species	To assess structural conservation of Tdp2 conformational changes between human and mouse Tdp2, we also determined a 3.2 Å resolution structure of the human Tdp2cat domain bound to a DNA 5′-PO4 terminus product complex (PDB entry 5INO).	RESULTS
156	163	Tdp2cat	structure_element	To assess structural conservation of Tdp2 conformational changes between human and mouse Tdp2, we also determined a 3.2 Å resolution structure of the human Tdp2cat domain bound to a DNA 5′-PO4 terminus product complex (PDB entry 5INO).	RESULTS
171	179	bound to	protein_state	To assess structural conservation of Tdp2 conformational changes between human and mouse Tdp2, we also determined a 3.2 Å resolution structure of the human Tdp2cat domain bound to a DNA 5′-PO4 terminus product complex (PDB entry 5INO).	RESULTS
182	192	DNA 5′-PO4	chemical	To assess structural conservation of Tdp2 conformational changes between human and mouse Tdp2, we also determined a 3.2 Å resolution structure of the human Tdp2cat domain bound to a DNA 5′-PO4 terminus product complex (PDB entry 5INO).	RESULTS
19	24	human	species	Comparisons of the human hTdp2cat-DNA complex structure to the mTdp2cat DNA bound state show a high level of conservation of the DNA-bound conformations (Supplementary Figure S3A).	RESULTS
25	37	hTdp2cat-DNA	complex_assembly	Comparisons of the human hTdp2cat-DNA complex structure to the mTdp2cat DNA bound state show a high level of conservation of the DNA-bound conformations (Supplementary Figure S3A).	RESULTS
46	55	structure	evidence	Comparisons of the human hTdp2cat-DNA complex structure to the mTdp2cat DNA bound state show a high level of conservation of the DNA-bound conformations (Supplementary Figure S3A).	RESULTS
63	71	mTdp2cat	structure_element	Comparisons of the human hTdp2cat-DNA complex structure to the mTdp2cat DNA bound state show a high level of conservation of the DNA-bound conformations (Supplementary Figure S3A).	RESULTS
72	81	DNA bound	protein_state	Comparisons of the human hTdp2cat-DNA complex structure to the mTdp2cat DNA bound state show a high level of conservation of the DNA-bound conformations (Supplementary Figure S3A).	RESULTS
129	138	DNA-bound	protein_state	Comparisons of the human hTdp2cat-DNA complex structure to the mTdp2cat DNA bound state show a high level of conservation of the DNA-bound conformations (Supplementary Figure S3A).	RESULTS
21	29	mTdp2cat	structure_element	Moreover, similar to mTdp2cat, proteolytic protection of the hTdp2cat substrate binding loop occurs with addition of Mg2+ and DNA (Supplementary Figure S3B).	RESULTS
61	69	hTdp2cat	structure_element	Moreover, similar to mTdp2cat, proteolytic protection of the hTdp2cat substrate binding loop occurs with addition of Mg2+ and DNA (Supplementary Figure S3B).	RESULTS
70	92	substrate binding loop	structure_element	Moreover, similar to mTdp2cat, proteolytic protection of the hTdp2cat substrate binding loop occurs with addition of Mg2+ and DNA (Supplementary Figure S3B).	RESULTS
117	121	Mg2+	chemical	Moreover, similar to mTdp2cat, proteolytic protection of the hTdp2cat substrate binding loop occurs with addition of Mg2+ and DNA (Supplementary Figure S3B).	RESULTS
126	129	DNA	chemical	Moreover, similar to mTdp2cat, proteolytic protection of the hTdp2cat substrate binding loop occurs with addition of Mg2+ and DNA (Supplementary Figure S3B).	RESULTS
6	11	X-ray	experimental_method	Thus, X-ray structures and limited proteolysis analysis indicate that DNA- and metal-induced conformational changes are a conserved feature of the vertebrate Tdp2-substrate interaction.	RESULTS
12	22	structures	evidence	Thus, X-ray structures and limited proteolysis analysis indicate that DNA- and metal-induced conformational changes are a conserved feature of the vertebrate Tdp2-substrate interaction.	RESULTS
27	55	limited proteolysis analysis	experimental_method	Thus, X-ray structures and limited proteolysis analysis indicate that DNA- and metal-induced conformational changes are a conserved feature of the vertebrate Tdp2-substrate interaction.	RESULTS
70	73	DNA	chemical	Thus, X-ray structures and limited proteolysis analysis indicate that DNA- and metal-induced conformational changes are a conserved feature of the vertebrate Tdp2-substrate interaction.	RESULTS
122	131	conserved	protein_state	Thus, X-ray structures and limited proteolysis analysis indicate that DNA- and metal-induced conformational changes are a conserved feature of the vertebrate Tdp2-substrate interaction.	RESULTS
147	157	vertebrate	taxonomy_domain	Thus, X-ray structures and limited proteolysis analysis indicate that DNA- and metal-induced conformational changes are a conserved feature of the vertebrate Tdp2-substrate interaction.	RESULTS
158	162	Tdp2	protein	Thus, X-ray structures and limited proteolysis analysis indicate that DNA- and metal-induced conformational changes are a conserved feature of the vertebrate Tdp2-substrate interaction.	RESULTS
0	4	Tdp2	protein	Tdp2 metal ion dependence	RESULTS
32	57	X-ray structural analyses	experimental_method	Consistently in high-resolution X-ray structural analyses we, and others observe a single Mg2+ metal bound in the Tdp2 active site.	RESULTS
90	94	Mg2+	chemical	Consistently in high-resolution X-ray structural analyses we, and others observe a single Mg2+ metal bound in the Tdp2 active site.	RESULTS
101	109	bound in	protein_state	Consistently in high-resolution X-ray structural analyses we, and others observe a single Mg2+ metal bound in the Tdp2 active site.	RESULTS
114	118	Tdp2	protein	Consistently in high-resolution X-ray structural analyses we, and others observe a single Mg2+ metal bound in the Tdp2 active site.	RESULTS
119	130	active site	site	Consistently in high-resolution X-ray structural analyses we, and others observe a single Mg2+ metal bound in the Tdp2 active site.	RESULTS
18	26	DNA-free	protein_state	This includes the DNA-free (Figure 3A), DNA damage bound (Figure 3B) and reaction product-bound crystal forms of mouse, (PDB entry 4GZ1), D. rerio (PDB entry 4FPV) and C. elegans Tdp2 (PDB entry 4FVA).	RESULTS
40	56	DNA damage bound	protein_state	This includes the DNA-free (Figure 3A), DNA damage bound (Figure 3B) and reaction product-bound crystal forms of mouse, (PDB entry 4GZ1), D. rerio (PDB entry 4FPV) and C. elegans Tdp2 (PDB entry 4FVA).	RESULTS
73	95	reaction product-bound	protein_state	This includes the DNA-free (Figure 3A), DNA damage bound (Figure 3B) and reaction product-bound crystal forms of mouse, (PDB entry 4GZ1), D. rerio (PDB entry 4FPV) and C. elegans Tdp2 (PDB entry 4FVA).	RESULTS
96	109	crystal forms	evidence	This includes the DNA-free (Figure 3A), DNA damage bound (Figure 3B) and reaction product-bound crystal forms of mouse, (PDB entry 4GZ1), D. rerio (PDB entry 4FPV) and C. elegans Tdp2 (PDB entry 4FVA).	RESULTS
113	118	mouse	taxonomy_domain	This includes the DNA-free (Figure 3A), DNA damage bound (Figure 3B) and reaction product-bound crystal forms of mouse, (PDB entry 4GZ1), D. rerio (PDB entry 4FPV) and C. elegans Tdp2 (PDB entry 4FVA).	RESULTS
138	146	D. rerio	species	This includes the DNA-free (Figure 3A), DNA damage bound (Figure 3B) and reaction product-bound crystal forms of mouse, (PDB entry 4GZ1), D. rerio (PDB entry 4FPV) and C. elegans Tdp2 (PDB entry 4FVA).	RESULTS
168	178	C. elegans	species	This includes the DNA-free (Figure 3A), DNA damage bound (Figure 3B) and reaction product-bound crystal forms of mouse, (PDB entry 4GZ1), D. rerio (PDB entry 4FPV) and C. elegans Tdp2 (PDB entry 4FVA).	RESULTS
179	183	Tdp2	protein	This includes the DNA-free (Figure 3A), DNA damage bound (Figure 3B) and reaction product-bound crystal forms of mouse, (PDB entry 4GZ1), D. rerio (PDB entry 4FPV) and C. elegans Tdp2 (PDB entry 4FVA).	RESULTS
18	38	biochemical analysis	experimental_method	However, previous biochemical analysis has suggested an alternative two-metal ion mechanism for the Tdp2-phosphotyrosyl phosphodiesterase reaction.	RESULTS
100	104	Tdp2	protein	However, previous biochemical analysis has suggested an alternative two-metal ion mechanism for the Tdp2-phosphotyrosyl phosphodiesterase reaction.	RESULTS
105	137	phosphotyrosyl phosphodiesterase	protein_type	However, previous biochemical analysis has suggested an alternative two-metal ion mechanism for the Tdp2-phosphotyrosyl phosphodiesterase reaction.	RESULTS
34	38	Mg2+	chemical	In these experiments, at limiting Mg2+ concentrations, Ca2+ addition to Tdp2 reactions stimulated activity.	RESULTS
55	59	Ca2+	chemical	In these experiments, at limiting Mg2+ concentrations, Ca2+ addition to Tdp2 reactions stimulated activity.	RESULTS
72	76	Tdp2	protein	In these experiments, at limiting Mg2+ concentrations, Ca2+ addition to Tdp2 reactions stimulated activity.	RESULTS
64	78	phosphotyrosyl	ptm	While this work was suggestive of a two metal ion mechanism for phosphotyrosyl bond cleavage by Tdp2, we note that second metal ion titrations can be influenced by metal ion binding sites outside of the active site.	RESULTS
96	100	Tdp2	protein	While this work was suggestive of a two metal ion mechanism for phosphotyrosyl bond cleavage by Tdp2, we note that second metal ion titrations can be influenced by metal ion binding sites outside of the active site.	RESULTS
164	187	metal ion binding sites	site	While this work was suggestive of a two metal ion mechanism for phosphotyrosyl bond cleavage by Tdp2, we note that second metal ion titrations can be influenced by metal ion binding sites outside of the active site.	RESULTS
203	214	active site	site	While this work was suggestive of a two metal ion mechanism for phosphotyrosyl bond cleavage by Tdp2, we note that second metal ion titrations can be influenced by metal ion binding sites outside of the active site.	RESULTS
9	24	divalent metals	chemical	In fact, divalent metals have been observed in the Tdp2 protein–DNA complexes (PDB entry 4GZ2) distal to the active center, and we propose this might account for varied results in different studies.	RESULTS
51	55	Tdp2	protein	In fact, divalent metals have been observed in the Tdp2 protein–DNA complexes (PDB entry 4GZ2) distal to the active center, and we propose this might account for varied results in different studies.	RESULTS
64	67	DNA	chemical	In fact, divalent metals have been observed in the Tdp2 protein–DNA complexes (PDB entry 4GZ2) distal to the active center, and we propose this might account for varied results in different studies.	RESULTS
109	122	active center	site	In fact, divalent metals have been observed in the Tdp2 protein–DNA complexes (PDB entry 4GZ2) distal to the active center, and we propose this might account for varied results in different studies.	RESULTS
49	53	Tdp2	protein	To further probe the metal ion dependence of the Tdp2 phosphodiesterase reaction, we performed metal ion binding assays, determined crystal structures in the presence of varied divalent metals (Mn2+ and Ca2+), and analyzed metal ion dependence of the Tdp2 phosphotyrosyl phosphodiesterase reaction (Figure 4).	RESULTS
54	71	phosphodiesterase	protein_type	To further probe the metal ion dependence of the Tdp2 phosphodiesterase reaction, we performed metal ion binding assays, determined crystal structures in the presence of varied divalent metals (Mn2+ and Ca2+), and analyzed metal ion dependence of the Tdp2 phosphotyrosyl phosphodiesterase reaction (Figure 4).	RESULTS
95	119	metal ion binding assays	experimental_method	To further probe the metal ion dependence of the Tdp2 phosphodiesterase reaction, we performed metal ion binding assays, determined crystal structures in the presence of varied divalent metals (Mn2+ and Ca2+), and analyzed metal ion dependence of the Tdp2 phosphotyrosyl phosphodiesterase reaction (Figure 4).	RESULTS
132	150	crystal structures	evidence	To further probe the metal ion dependence of the Tdp2 phosphodiesterase reaction, we performed metal ion binding assays, determined crystal structures in the presence of varied divalent metals (Mn2+ and Ca2+), and analyzed metal ion dependence of the Tdp2 phosphotyrosyl phosphodiesterase reaction (Figure 4).	RESULTS
158	169	presence of	protein_state	To further probe the metal ion dependence of the Tdp2 phosphodiesterase reaction, we performed metal ion binding assays, determined crystal structures in the presence of varied divalent metals (Mn2+ and Ca2+), and analyzed metal ion dependence of the Tdp2 phosphotyrosyl phosphodiesterase reaction (Figure 4).	RESULTS
177	192	divalent metals	chemical	To further probe the metal ion dependence of the Tdp2 phosphodiesterase reaction, we performed metal ion binding assays, determined crystal structures in the presence of varied divalent metals (Mn2+ and Ca2+), and analyzed metal ion dependence of the Tdp2 phosphotyrosyl phosphodiesterase reaction (Figure 4).	RESULTS
194	198	Mn2+	chemical	To further probe the metal ion dependence of the Tdp2 phosphodiesterase reaction, we performed metal ion binding assays, determined crystal structures in the presence of varied divalent metals (Mn2+ and Ca2+), and analyzed metal ion dependence of the Tdp2 phosphotyrosyl phosphodiesterase reaction (Figure 4).	RESULTS
203	208	Ca2+)	chemical	To further probe the metal ion dependence of the Tdp2 phosphodiesterase reaction, we performed metal ion binding assays, determined crystal structures in the presence of varied divalent metals (Mn2+ and Ca2+), and analyzed metal ion dependence of the Tdp2 phosphotyrosyl phosphodiesterase reaction (Figure 4).	RESULTS
251	255	Tdp2	protein	To further probe the metal ion dependence of the Tdp2 phosphodiesterase reaction, we performed metal ion binding assays, determined crystal structures in the presence of varied divalent metals (Mn2+ and Ca2+), and analyzed metal ion dependence of the Tdp2 phosphotyrosyl phosphodiesterase reaction (Figure 4).	RESULTS
256	288	phosphotyrosyl phosphodiesterase	protein_type	To further probe the metal ion dependence of the Tdp2 phosphodiesterase reaction, we performed metal ion binding assays, determined crystal structures in the presence of varied divalent metals (Mn2+ and Ca2+), and analyzed metal ion dependence of the Tdp2 phosphotyrosyl phosphodiesterase reaction (Figure 4).	RESULTS
33	37	Tdp2	protein	Metal cofactor interactions with Tdp2. (A) Intrinsic tryptophan fluorescence of mTdp2cat was used to monitor a conformational response to divalent metal ion binding.	FIG
43	76	Intrinsic tryptophan fluorescence	evidence	Metal cofactor interactions with Tdp2. (A) Intrinsic tryptophan fluorescence of mTdp2cat was used to monitor a conformational response to divalent metal ion binding.	FIG
80	88	mTdp2cat	structure_element	Metal cofactor interactions with Tdp2. (A) Intrinsic tryptophan fluorescence of mTdp2cat was used to monitor a conformational response to divalent metal ion binding.	FIG
7	11	Mg2+	chemical	Either Mg2+ or Ca2+ were titrated in the presence or absence of 5′-P DNA, and the tryptophan fluorescence was monitored with an excitation wavelength of 280 nm and emission wavelength of 350 nm using 10 nm band pass filters.	FIG
15	19	Ca2+	chemical	Either Mg2+ or Ca2+ were titrated in the presence or absence of 5′-P DNA, and the tryptophan fluorescence was monitored with an excitation wavelength of 280 nm and emission wavelength of 350 nm using 10 nm band pass filters.	FIG
25	33	titrated	experimental_method	Either Mg2+ or Ca2+ were titrated in the presence or absence of 5′-P DNA, and the tryptophan fluorescence was monitored with an excitation wavelength of 280 nm and emission wavelength of 350 nm using 10 nm band pass filters.	FIG
41	49	presence	protein_state	Either Mg2+ or Ca2+ were titrated in the presence or absence of 5′-P DNA, and the tryptophan fluorescence was monitored with an excitation wavelength of 280 nm and emission wavelength of 350 nm using 10 nm band pass filters.	FIG
53	63	absence of	protein_state	Either Mg2+ or Ca2+ were titrated in the presence or absence of 5′-P DNA, and the tryptophan fluorescence was monitored with an excitation wavelength of 280 nm and emission wavelength of 350 nm using 10 nm band pass filters.	FIG
64	72	5′-P DNA	chemical	Either Mg2+ or Ca2+ were titrated in the presence or absence of 5′-P DNA, and the tryptophan fluorescence was monitored with an excitation wavelength of 280 nm and emission wavelength of 350 nm using 10 nm band pass filters.	FIG
82	105	tryptophan fluorescence	evidence	Either Mg2+ or Ca2+ were titrated in the presence or absence of 5′-P DNA, and the tryptophan fluorescence was monitored with an excitation wavelength of 280 nm and emission wavelength of 350 nm using 10 nm band pass filters.	FIG
5	9	Mg2+	chemical	Both Mg2+ and Ca2+ induce a conformational change which elicits an increase in tryptophan fluorescence of mTdp2cat in the presence and absence of DNA, while D358N active site mutant of mTdp2cat is unresponsive to Mg2+. (B) mTdp2cat activity assayed on a T5PNP substrate as a function of Mg2+ and Ca2+ concentration.	FIG
14	18	Ca2+	chemical	Both Mg2+ and Ca2+ induce a conformational change which elicits an increase in tryptophan fluorescence of mTdp2cat in the presence and absence of DNA, while D358N active site mutant of mTdp2cat is unresponsive to Mg2+. (B) mTdp2cat activity assayed on a T5PNP substrate as a function of Mg2+ and Ca2+ concentration.	FIG
79	102	tryptophan fluorescence	evidence	Both Mg2+ and Ca2+ induce a conformational change which elicits an increase in tryptophan fluorescence of mTdp2cat in the presence and absence of DNA, while D358N active site mutant of mTdp2cat is unresponsive to Mg2+. (B) mTdp2cat activity assayed on a T5PNP substrate as a function of Mg2+ and Ca2+ concentration.	FIG
106	114	mTdp2cat	structure_element	Both Mg2+ and Ca2+ induce a conformational change which elicits an increase in tryptophan fluorescence of mTdp2cat in the presence and absence of DNA, while D358N active site mutant of mTdp2cat is unresponsive to Mg2+. (B) mTdp2cat activity assayed on a T5PNP substrate as a function of Mg2+ and Ca2+ concentration.	FIG
122	130	presence	protein_state	Both Mg2+ and Ca2+ induce a conformational change which elicits an increase in tryptophan fluorescence of mTdp2cat in the presence and absence of DNA, while D358N active site mutant of mTdp2cat is unresponsive to Mg2+. (B) mTdp2cat activity assayed on a T5PNP substrate as a function of Mg2+ and Ca2+ concentration.	FIG
135	145	absence of	protein_state	Both Mg2+ and Ca2+ induce a conformational change which elicits an increase in tryptophan fluorescence of mTdp2cat in the presence and absence of DNA, while D358N active site mutant of mTdp2cat is unresponsive to Mg2+. (B) mTdp2cat activity assayed on a T5PNP substrate as a function of Mg2+ and Ca2+ concentration.	FIG
146	149	DNA	chemical	Both Mg2+ and Ca2+ induce a conformational change which elicits an increase in tryptophan fluorescence of mTdp2cat in the presence and absence of DNA, while D358N active site mutant of mTdp2cat is unresponsive to Mg2+. (B) mTdp2cat activity assayed on a T5PNP substrate as a function of Mg2+ and Ca2+ concentration.	FIG
157	162	D358N	mutant	Both Mg2+ and Ca2+ induce a conformational change which elicits an increase in tryptophan fluorescence of mTdp2cat in the presence and absence of DNA, while D358N active site mutant of mTdp2cat is unresponsive to Mg2+. (B) mTdp2cat activity assayed on a T5PNP substrate as a function of Mg2+ and Ca2+ concentration.	FIG
163	174	active site	site	Both Mg2+ and Ca2+ induce a conformational change which elicits an increase in tryptophan fluorescence of mTdp2cat in the presence and absence of DNA, while D358N active site mutant of mTdp2cat is unresponsive to Mg2+. (B) mTdp2cat activity assayed on a T5PNP substrate as a function of Mg2+ and Ca2+ concentration.	FIG
175	181	mutant	protein_state	Both Mg2+ and Ca2+ induce a conformational change which elicits an increase in tryptophan fluorescence of mTdp2cat in the presence and absence of DNA, while D358N active site mutant of mTdp2cat is unresponsive to Mg2+. (B) mTdp2cat activity assayed on a T5PNP substrate as a function of Mg2+ and Ca2+ concentration.	FIG
185	193	mTdp2cat	structure_element	Both Mg2+ and Ca2+ induce a conformational change which elicits an increase in tryptophan fluorescence of mTdp2cat in the presence and absence of DNA, while D358N active site mutant of mTdp2cat is unresponsive to Mg2+. (B) mTdp2cat activity assayed on a T5PNP substrate as a function of Mg2+ and Ca2+ concentration.	FIG
197	209	unresponsive	protein_state	Both Mg2+ and Ca2+ induce a conformational change which elicits an increase in tryptophan fluorescence of mTdp2cat in the presence and absence of DNA, while D358N active site mutant of mTdp2cat is unresponsive to Mg2+. (B) mTdp2cat activity assayed on a T5PNP substrate as a function of Mg2+ and Ca2+ concentration.	FIG
213	217	Mg2+	chemical	Both Mg2+ and Ca2+ induce a conformational change which elicits an increase in tryptophan fluorescence of mTdp2cat in the presence and absence of DNA, while D358N active site mutant of mTdp2cat is unresponsive to Mg2+. (B) mTdp2cat activity assayed on a T5PNP substrate as a function of Mg2+ and Ca2+ concentration.	FIG
223	231	mTdp2cat	structure_element	Both Mg2+ and Ca2+ induce a conformational change which elicits an increase in tryptophan fluorescence of mTdp2cat in the presence and absence of DNA, while D358N active site mutant of mTdp2cat is unresponsive to Mg2+. (B) mTdp2cat activity assayed on a T5PNP substrate as a function of Mg2+ and Ca2+ concentration.	FIG
254	259	T5PNP	chemical	Both Mg2+ and Ca2+ induce a conformational change which elicits an increase in tryptophan fluorescence of mTdp2cat in the presence and absence of DNA, while D358N active site mutant of mTdp2cat is unresponsive to Mg2+. (B) mTdp2cat activity assayed on a T5PNP substrate as a function of Mg2+ and Ca2+ concentration.	FIG
287	291	Mg2+	chemical	Both Mg2+ and Ca2+ induce a conformational change which elicits an increase in tryptophan fluorescence of mTdp2cat in the presence and absence of DNA, while D358N active site mutant of mTdp2cat is unresponsive to Mg2+. (B) mTdp2cat activity assayed on a T5PNP substrate as a function of Mg2+ and Ca2+ concentration.	FIG
296	300	Ca2+	chemical	Both Mg2+ and Ca2+ induce a conformational change which elicits an increase in tryptophan fluorescence of mTdp2cat in the presence and absence of DNA, while D358N active site mutant of mTdp2cat is unresponsive to Mg2+. (B) mTdp2cat activity assayed on a T5PNP substrate as a function of Mg2+ and Ca2+ concentration.	FIG
0	3	PNP	chemical	PNP release (monitored by absorbance at 415 nm) as a function of Mg2+ concentration and in the absence or presence of 1 or 10 mM Ca2+ is shown; error bars, s.d. n = 4. (C) σ-A weighted 2Fo-Fc electron density map (blue) and model-phased anomalous difference Fourier (magenta) maps for the mTdp2cat–DNA–Mn2+ complex (PDB entry 5INP) show a single Mn2+ (cyan) is bound with expected octahedral coordination geometry.	FIG
65	69	Mg2+	chemical	PNP release (monitored by absorbance at 415 nm) as a function of Mg2+ concentration and in the absence or presence of 1 or 10 mM Ca2+ is shown; error bars, s.d. n = 4. (C) σ-A weighted 2Fo-Fc electron density map (blue) and model-phased anomalous difference Fourier (magenta) maps for the mTdp2cat–DNA–Mn2+ complex (PDB entry 5INP) show a single Mn2+ (cyan) is bound with expected octahedral coordination geometry.	FIG
95	102	absence	protein_state	PNP release (monitored by absorbance at 415 nm) as a function of Mg2+ concentration and in the absence or presence of 1 or 10 mM Ca2+ is shown; error bars, s.d. n = 4. (C) σ-A weighted 2Fo-Fc electron density map (blue) and model-phased anomalous difference Fourier (magenta) maps for the mTdp2cat–DNA–Mn2+ complex (PDB entry 5INP) show a single Mn2+ (cyan) is bound with expected octahedral coordination geometry.	FIG
106	117	presence of	protein_state	PNP release (monitored by absorbance at 415 nm) as a function of Mg2+ concentration and in the absence or presence of 1 or 10 mM Ca2+ is shown; error bars, s.d. n = 4. (C) σ-A weighted 2Fo-Fc electron density map (blue) and model-phased anomalous difference Fourier (magenta) maps for the mTdp2cat–DNA–Mn2+ complex (PDB entry 5INP) show a single Mn2+ (cyan) is bound with expected octahedral coordination geometry.	FIG
129	133	Ca2+	chemical	PNP release (monitored by absorbance at 415 nm) as a function of Mg2+ concentration and in the absence or presence of 1 or 10 mM Ca2+ is shown; error bars, s.d. n = 4. (C) σ-A weighted 2Fo-Fc electron density map (blue) and model-phased anomalous difference Fourier (magenta) maps for the mTdp2cat–DNA–Mn2+ complex (PDB entry 5INP) show a single Mn2+ (cyan) is bound with expected octahedral coordination geometry.	FIG
172	212	σ-A weighted 2Fo-Fc electron density map	evidence	PNP release (monitored by absorbance at 415 nm) as a function of Mg2+ concentration and in the absence or presence of 1 or 10 mM Ca2+ is shown; error bars, s.d. n = 4. (C) σ-A weighted 2Fo-Fc electron density map (blue) and model-phased anomalous difference Fourier (magenta) maps for the mTdp2cat–DNA–Mn2+ complex (PDB entry 5INP) show a single Mn2+ (cyan) is bound with expected octahedral coordination geometry.	FIG
224	265	model-phased anomalous difference Fourier	evidence	PNP release (monitored by absorbance at 415 nm) as a function of Mg2+ concentration and in the absence or presence of 1 or 10 mM Ca2+ is shown; error bars, s.d. n = 4. (C) σ-A weighted 2Fo-Fc electron density map (blue) and model-phased anomalous difference Fourier (magenta) maps for the mTdp2cat–DNA–Mn2+ complex (PDB entry 5INP) show a single Mn2+ (cyan) is bound with expected octahedral coordination geometry.	FIG
276	280	maps	evidence	PNP release (monitored by absorbance at 415 nm) as a function of Mg2+ concentration and in the absence or presence of 1 or 10 mM Ca2+ is shown; error bars, s.d. n = 4. (C) σ-A weighted 2Fo-Fc electron density map (blue) and model-phased anomalous difference Fourier (magenta) maps for the mTdp2cat–DNA–Mn2+ complex (PDB entry 5INP) show a single Mn2+ (cyan) is bound with expected octahedral coordination geometry.	FIG
289	306	mTdp2cat–DNA–Mn2+	complex_assembly	PNP release (monitored by absorbance at 415 nm) as a function of Mg2+ concentration and in the absence or presence of 1 or 10 mM Ca2+ is shown; error bars, s.d. n = 4. (C) σ-A weighted 2Fo-Fc electron density map (blue) and model-phased anomalous difference Fourier (magenta) maps for the mTdp2cat–DNA–Mn2+ complex (PDB entry 5INP) show a single Mn2+ (cyan) is bound with expected octahedral coordination geometry.	FIG
346	350	Mn2+	chemical	PNP release (monitored by absorbance at 415 nm) as a function of Mg2+ concentration and in the absence or presence of 1 or 10 mM Ca2+ is shown; error bars, s.d. n = 4. (C) σ-A weighted 2Fo-Fc electron density map (blue) and model-phased anomalous difference Fourier (magenta) maps for the mTdp2cat–DNA–Mn2+ complex (PDB entry 5INP) show a single Mn2+ (cyan) is bound with expected octahedral coordination geometry.	FIG
18	50	anomalous difference Fourier map	evidence	A 53σ peak in the anomalous difference Fourier map (data collected at λ = 1.5418 Å) supports Mn2+ as the identity of this atom.	FIG
93	97	Mn2+	chemical	A 53σ peak in the anomalous difference Fourier map (data collected at λ = 1.5418 Å) supports Mn2+ as the identity of this atom.	FIG
18	22	Ca2+	chemical	(D) Comparison of Ca2+ (green Ca2+ ion, orange DNA) (PDB entry 5INQ), and Mg2+ (magenta Mg2+ ion, yellow DNA) (PDB entry 4GZ1) mTdp2cat–DNA structures shows that Ca2+ distorts the 5′-phosphate binding mode.	FIG
30	34	Ca2+	chemical	(D) Comparison of Ca2+ (green Ca2+ ion, orange DNA) (PDB entry 5INQ), and Mg2+ (magenta Mg2+ ion, yellow DNA) (PDB entry 4GZ1) mTdp2cat–DNA structures shows that Ca2+ distorts the 5′-phosphate binding mode.	FIG
47	50	DNA	chemical	(D) Comparison of Ca2+ (green Ca2+ ion, orange DNA) (PDB entry 5INQ), and Mg2+ (magenta Mg2+ ion, yellow DNA) (PDB entry 4GZ1) mTdp2cat–DNA structures shows that Ca2+ distorts the 5′-phosphate binding mode.	FIG
74	78	Mg2+	chemical	(D) Comparison of Ca2+ (green Ca2+ ion, orange DNA) (PDB entry 5INQ), and Mg2+ (magenta Mg2+ ion, yellow DNA) (PDB entry 4GZ1) mTdp2cat–DNA structures shows that Ca2+ distorts the 5′-phosphate binding mode.	FIG
88	92	Mg2+	chemical	(D) Comparison of Ca2+ (green Ca2+ ion, orange DNA) (PDB entry 5INQ), and Mg2+ (magenta Mg2+ ion, yellow DNA) (PDB entry 4GZ1) mTdp2cat–DNA structures shows that Ca2+ distorts the 5′-phosphate binding mode.	FIG
105	108	DNA	chemical	(D) Comparison of Ca2+ (green Ca2+ ion, orange DNA) (PDB entry 5INQ), and Mg2+ (magenta Mg2+ ion, yellow DNA) (PDB entry 4GZ1) mTdp2cat–DNA structures shows that Ca2+ distorts the 5′-phosphate binding mode.	FIG
127	139	mTdp2cat–DNA	complex_assembly	(D) Comparison of Ca2+ (green Ca2+ ion, orange DNA) (PDB entry 5INQ), and Mg2+ (magenta Mg2+ ion, yellow DNA) (PDB entry 4GZ1) mTdp2cat–DNA structures shows that Ca2+ distorts the 5′-phosphate binding mode.	FIG
140	150	structures	evidence	(D) Comparison of Ca2+ (green Ca2+ ion, orange DNA) (PDB entry 5INQ), and Mg2+ (magenta Mg2+ ion, yellow DNA) (PDB entry 4GZ1) mTdp2cat–DNA structures shows that Ca2+ distorts the 5′-phosphate binding mode.	FIG
162	166	Ca2+	chemical	(D) Comparison of Ca2+ (green Ca2+ ion, orange DNA) (PDB entry 5INQ), and Mg2+ (magenta Mg2+ ion, yellow DNA) (PDB entry 4GZ1) mTdp2cat–DNA structures shows that Ca2+ distorts the 5′-phosphate binding mode.	FIG
180	205	5′-phosphate binding mode	site	(D) Comparison of Ca2+ (green Ca2+ ion, orange DNA) (PDB entry 5INQ), and Mg2+ (magenta Mg2+ ion, yellow DNA) (PDB entry 4GZ1) mTdp2cat–DNA structures shows that Ca2+ distorts the 5′-phosphate binding mode.	FIG
4	15	proteolysis	experimental_method	Our proteolysis results indicate a Mg2+-dependent Tdp2 conformational response to metal binding.	RESULTS
35	39	Mg2+	chemical	Our proteolysis results indicate a Mg2+-dependent Tdp2 conformational response to metal binding.	RESULTS
50	54	Tdp2	protein	Our proteolysis results indicate a Mg2+-dependent Tdp2 conformational response to metal binding.	RESULTS
4	8	Tdp2	protein	The Tdp2 active site has three tryptophan residues within 10 Å of the metal binding center, so we assayed intrinsic tryptophan fluorescence to detect metal-induced conformational changes in mTdp2cat.	RESULTS
9	20	active site	site	The Tdp2 active site has three tryptophan residues within 10 Å of the metal binding center, so we assayed intrinsic tryptophan fluorescence to detect metal-induced conformational changes in mTdp2cat.	RESULTS
31	41	tryptophan	residue_name	The Tdp2 active site has three tryptophan residues within 10 Å of the metal binding center, so we assayed intrinsic tryptophan fluorescence to detect metal-induced conformational changes in mTdp2cat.	RESULTS
70	90	metal binding center	site	The Tdp2 active site has three tryptophan residues within 10 Å of the metal binding center, so we assayed intrinsic tryptophan fluorescence to detect metal-induced conformational changes in mTdp2cat.	RESULTS
106	139	intrinsic tryptophan fluorescence	evidence	The Tdp2 active site has three tryptophan residues within 10 Å of the metal binding center, so we assayed intrinsic tryptophan fluorescence to detect metal-induced conformational changes in mTdp2cat.	RESULTS
190	198	mTdp2cat	structure_element	The Tdp2 active site has three tryptophan residues within 10 Å of the metal binding center, so we assayed intrinsic tryptophan fluorescence to detect metal-induced conformational changes in mTdp2cat.	RESULTS
76	84	presence	protein_state	These data were an excellent fit to a single-site binding model both in the presence and absence of DNA (Figure 4A).	RESULTS
89	99	absence of	protein_state	These data were an excellent fit to a single-site binding model both in the presence and absence of DNA (Figure 4A).	RESULTS
100	103	DNA	chemical	These data were an excellent fit to a single-site binding model both in the presence and absence of DNA (Figure 4A).	RESULTS
23	27	Mg2+	chemical	This analysis revealed Mg2+ Kd values in the sub-millimolar range and Hill coefficients which were consistent with a single metal binding site both in the presence and absence of DNA (Supplementary Table S2).	RESULTS
28	30	Kd	evidence	This analysis revealed Mg2+ Kd values in the sub-millimolar range and Hill coefficients which were consistent with a single metal binding site both in the presence and absence of DNA (Supplementary Table S2).	RESULTS
70	87	Hill coefficients	evidence	This analysis revealed Mg2+ Kd values in the sub-millimolar range and Hill coefficients which were consistent with a single metal binding site both in the presence and absence of DNA (Supplementary Table S2).	RESULTS
124	142	metal binding site	site	This analysis revealed Mg2+ Kd values in the sub-millimolar range and Hill coefficients which were consistent with a single metal binding site both in the presence and absence of DNA (Supplementary Table S2).	RESULTS
155	163	presence	protein_state	This analysis revealed Mg2+ Kd values in the sub-millimolar range and Hill coefficients which were consistent with a single metal binding site both in the presence and absence of DNA (Supplementary Table S2).	RESULTS
168	178	absence of	protein_state	This analysis revealed Mg2+ Kd values in the sub-millimolar range and Hill coefficients which were consistent with a single metal binding site both in the presence and absence of DNA (Supplementary Table S2).	RESULTS
179	182	DNA	chemical	This analysis revealed Mg2+ Kd values in the sub-millimolar range and Hill coefficients which were consistent with a single metal binding site both in the presence and absence of DNA (Supplementary Table S2).	RESULTS
56	60	Tdp2	protein	We then measured effects of metal ion concentrations on Tdp2 cleavage of p-nitrophenyl-thymidine-5′-phosphate by mTdp2cat.	RESULTS
73	109	p-nitrophenyl-thymidine-5′-phosphate	chemical	We then measured effects of metal ion concentrations on Tdp2 cleavage of p-nitrophenyl-thymidine-5′-phosphate by mTdp2cat.	RESULTS
113	121	mTdp2cat	structure_element	We then measured effects of metal ion concentrations on Tdp2 cleavage of p-nitrophenyl-thymidine-5′-phosphate by mTdp2cat.	RESULTS
72	75	DNA	chemical	This small molecule substrate is not expected to be influenced by metal–DNA coordination outside of the active site.	RESULTS
104	115	active site	site	This small molecule substrate is not expected to be influenced by metal–DNA coordination outside of the active site.	RESULTS
23	27	Ca2+	chemical	Inclusion of ultrapure Ca2+ (1 mM or 10 mM) results in a dose-dependent inhibition but not stimulation Tdp2 activity, even in conditions of limiting Mg2+ (Figure 4B).	RESULTS
103	107	Tdp2	protein	Inclusion of ultrapure Ca2+ (1 mM or 10 mM) results in a dose-dependent inhibition but not stimulation Tdp2 activity, even in conditions of limiting Mg2+ (Figure 4B).	RESULTS
149	153	Mg2+	chemical	Inclusion of ultrapure Ca2+ (1 mM or 10 mM) results in a dose-dependent inhibition but not stimulation Tdp2 activity, even in conditions of limiting Mg2+ (Figure 4B).	RESULTS
22	32	titrations	experimental_method	We performed the same titrations with human hTdp2FL and hTdp2cat (Supplementary Figure S4), and find similar stimulation of activity by Mg2+ and inhibition by Ca2+.	RESULTS
38	43	human	species	We performed the same titrations with human hTdp2FL and hTdp2cat (Supplementary Figure S4), and find similar stimulation of activity by Mg2+ and inhibition by Ca2+.	RESULTS
44	51	hTdp2FL	protein	We performed the same titrations with human hTdp2FL and hTdp2cat (Supplementary Figure S4), and find similar stimulation of activity by Mg2+ and inhibition by Ca2+.	RESULTS
56	64	hTdp2cat	structure_element	We performed the same titrations with human hTdp2FL and hTdp2cat (Supplementary Figure S4), and find similar stimulation of activity by Mg2+ and inhibition by Ca2+.	RESULTS
136	140	Mg2+	chemical	We performed the same titrations with human hTdp2FL and hTdp2cat (Supplementary Figure S4), and find similar stimulation of activity by Mg2+ and inhibition by Ca2+.	RESULTS
159	163	Ca2+	chemical	We performed the same titrations with human hTdp2FL and hTdp2cat (Supplementary Figure S4), and find similar stimulation of activity by Mg2+ and inhibition by Ca2+.	RESULTS
15	37	metal binding analyses	experimental_method	Overall, these metal binding analyses are consistent with a single metal ion mediated reaction.	RESULTS
72	76	Tdp2	protein	To further evaluate the structural influence of divalent cations on the Tdp2 active site, we determined crystal structures by soaking crystals with metal cofactors that either support (Mn2+) or inhibit (Ca2+, Figure 4B) the Tdp2 reaction (PDB entries 5INP and 5INQ).	RESULTS
77	88	active site	site	To further evaluate the structural influence of divalent cations on the Tdp2 active site, we determined crystal structures by soaking crystals with metal cofactors that either support (Mn2+) or inhibit (Ca2+, Figure 4B) the Tdp2 reaction (PDB entries 5INP and 5INQ).	RESULTS
104	122	crystal structures	evidence	To further evaluate the structural influence of divalent cations on the Tdp2 active site, we determined crystal structures by soaking crystals with metal cofactors that either support (Mn2+) or inhibit (Ca2+, Figure 4B) the Tdp2 reaction (PDB entries 5INP and 5INQ).	RESULTS
126	142	soaking crystals	experimental_method	To further evaluate the structural influence of divalent cations on the Tdp2 active site, we determined crystal structures by soaking crystals with metal cofactors that either support (Mn2+) or inhibit (Ca2+, Figure 4B) the Tdp2 reaction (PDB entries 5INP and 5INQ).	RESULTS
176	183	support	protein_state	To further evaluate the structural influence of divalent cations on the Tdp2 active site, we determined crystal structures by soaking crystals with metal cofactors that either support (Mn2+) or inhibit (Ca2+, Figure 4B) the Tdp2 reaction (PDB entries 5INP and 5INQ).	RESULTS
185	189	Mn2+	chemical	To further evaluate the structural influence of divalent cations on the Tdp2 active site, we determined crystal structures by soaking crystals with metal cofactors that either support (Mn2+) or inhibit (Ca2+, Figure 4B) the Tdp2 reaction (PDB entries 5INP and 5INQ).	RESULTS
194	201	inhibit	protein_state	To further evaluate the structural influence of divalent cations on the Tdp2 active site, we determined crystal structures by soaking crystals with metal cofactors that either support (Mn2+) or inhibit (Ca2+, Figure 4B) the Tdp2 reaction (PDB entries 5INP and 5INQ).	RESULTS
203	207	Ca2+	chemical	To further evaluate the structural influence of divalent cations on the Tdp2 active site, we determined crystal structures by soaking crystals with metal cofactors that either support (Mn2+) or inhibit (Ca2+, Figure 4B) the Tdp2 reaction (PDB entries 5INP and 5INQ).	RESULTS
224	228	Tdp2	protein	To further evaluate the structural influence of divalent cations on the Tdp2 active site, we determined crystal structures by soaking crystals with metal cofactors that either support (Mn2+) or inhibit (Ca2+, Figure 4B) the Tdp2 reaction (PDB entries 5INP and 5INQ).	RESULTS
0	33	Anomalous difference Fourier maps	evidence	Anomalous difference Fourier maps of the Tdp2–DNA–Mn2+ complex show a single binding site for Mn2+ in each Tdp2 active site (Figure 4C), with octahedral coordination and bond lengths typical for Mn2+ ligands (Supplementary Table S3).	RESULTS
41	54	Tdp2–DNA–Mn2+	complex_assembly	Anomalous difference Fourier maps of the Tdp2–DNA–Mn2+ complex show a single binding site for Mn2+ in each Tdp2 active site (Figure 4C), with octahedral coordination and bond lengths typical for Mn2+ ligands (Supplementary Table S3).	RESULTS
77	89	binding site	site	Anomalous difference Fourier maps of the Tdp2–DNA–Mn2+ complex show a single binding site for Mn2+ in each Tdp2 active site (Figure 4C), with octahedral coordination and bond lengths typical for Mn2+ ligands (Supplementary Table S3).	RESULTS
94	98	Mn2+	chemical	Anomalous difference Fourier maps of the Tdp2–DNA–Mn2+ complex show a single binding site for Mn2+ in each Tdp2 active site (Figure 4C), with octahedral coordination and bond lengths typical for Mn2+ ligands (Supplementary Table S3).	RESULTS
107	111	Tdp2	protein	Anomalous difference Fourier maps of the Tdp2–DNA–Mn2+ complex show a single binding site for Mn2+ in each Tdp2 active site (Figure 4C), with octahedral coordination and bond lengths typical for Mn2+ ligands (Supplementary Table S3).	RESULTS
112	123	active site	site	Anomalous difference Fourier maps of the Tdp2–DNA–Mn2+ complex show a single binding site for Mn2+ in each Tdp2 active site (Figure 4C), with octahedral coordination and bond lengths typical for Mn2+ ligands (Supplementary Table S3).	RESULTS
142	165	octahedral coordination	bond_interaction	Anomalous difference Fourier maps of the Tdp2–DNA–Mn2+ complex show a single binding site for Mn2+ in each Tdp2 active site (Figure 4C), with octahedral coordination and bond lengths typical for Mn2+ ligands (Supplementary Table S3).	RESULTS
195	199	Mn2+	chemical	Anomalous difference Fourier maps of the Tdp2–DNA–Mn2+ complex show a single binding site for Mn2+ in each Tdp2 active site (Figure 4C), with octahedral coordination and bond lengths typical for Mn2+ ligands (Supplementary Table S3).	RESULTS
4	8	Mn2+	chemical	The Mn2+ ion is positioned in the Tdp2 active site similar to the Mg2+-bound complex (Figure 2C), which is consistent with the ability of Mn2+ to support robust Tdp2 catalytic activity.	RESULTS
34	38	Tdp2	protein	The Mn2+ ion is positioned in the Tdp2 active site similar to the Mg2+-bound complex (Figure 2C), which is consistent with the ability of Mn2+ to support robust Tdp2 catalytic activity.	RESULTS
39	50	active site	site	The Mn2+ ion is positioned in the Tdp2 active site similar to the Mg2+-bound complex (Figure 2C), which is consistent with the ability of Mn2+ to support robust Tdp2 catalytic activity.	RESULTS
66	76	Mg2+-bound	protein_state	The Mn2+ ion is positioned in the Tdp2 active site similar to the Mg2+-bound complex (Figure 2C), which is consistent with the ability of Mn2+ to support robust Tdp2 catalytic activity.	RESULTS
138	142	Mn2+	chemical	The Mn2+ ion is positioned in the Tdp2 active site similar to the Mg2+-bound complex (Figure 2C), which is consistent with the ability of Mn2+ to support robust Tdp2 catalytic activity.	RESULTS
161	165	Tdp2	protein	The Mn2+ ion is positioned in the Tdp2 active site similar to the Mg2+-bound complex (Figure 2C), which is consistent with the ability of Mn2+ to support robust Tdp2 catalytic activity.	RESULTS
19	40	co-complex structures	evidence	In contrast, while co-complex structures with Ca2+ also show a single metal ion, Ca2+ binds in a slightly different position, shifted ∼1 Å from the Mg2+ site.	RESULTS
46	50	Ca2+	chemical	In contrast, while co-complex structures with Ca2+ also show a single metal ion, Ca2+ binds in a slightly different position, shifted ∼1 Å from the Mg2+ site.	RESULTS
81	85	Ca2+	chemical	In contrast, while co-complex structures with Ca2+ also show a single metal ion, Ca2+ binds in a slightly different position, shifted ∼1 Å from the Mg2+ site.	RESULTS
148	157	Mg2+ site	site	In contrast, while co-complex structures with Ca2+ also show a single metal ion, Ca2+ binds in a slightly different position, shifted ∼1 Å from the Mg2+ site.	RESULTS
9	13	Ca2+	chemical	Although Ca2+ is also octahedrally coordinated, longer bond lengths for the Ca2+ ligands (Supplementary Table S3) shift the Ca2+ ion relative to the Mg2+ ion site.	RESULTS
22	46	octahedrally coordinated	bond_interaction	Although Ca2+ is also octahedrally coordinated, longer bond lengths for the Ca2+ ligands (Supplementary Table S3) shift the Ca2+ ion relative to the Mg2+ ion site.	RESULTS
76	80	Ca2+	chemical	Although Ca2+ is also octahedrally coordinated, longer bond lengths for the Ca2+ ligands (Supplementary Table S3) shift the Ca2+ ion relative to the Mg2+ ion site.	RESULTS
124	128	Ca2+	chemical	Although Ca2+ is also octahedrally coordinated, longer bond lengths for the Ca2+ ligands (Supplementary Table S3) shift the Ca2+ ion relative to the Mg2+ ion site.	RESULTS
149	162	Mg2+ ion site	site	Although Ca2+ is also octahedrally coordinated, longer bond lengths for the Ca2+ ligands (Supplementary Table S3) shift the Ca2+ ion relative to the Mg2+ ion site.	RESULTS
15	53	bi-dentate inner sphere metal contacts	bond_interaction	Interestingly, bi-dentate inner sphere metal contacts from the Ca2+ ion to Glu162 distort the active site phosphate-binding mode, and dislodge the 5′-PO4 out of the Tdp2 active site (Figure 4D).	RESULTS
63	67	Ca2+	chemical	Interestingly, bi-dentate inner sphere metal contacts from the Ca2+ ion to Glu162 distort the active site phosphate-binding mode, and dislodge the 5′-PO4 out of the Tdp2 active site (Figure 4D).	RESULTS
75	81	Glu162	residue_name_number	Interestingly, bi-dentate inner sphere metal contacts from the Ca2+ ion to Glu162 distort the active site phosphate-binding mode, and dislodge the 5′-PO4 out of the Tdp2 active site (Figure 4D).	RESULTS
94	128	active site phosphate-binding mode	site	Interestingly, bi-dentate inner sphere metal contacts from the Ca2+ ion to Glu162 distort the active site phosphate-binding mode, and dislodge the 5′-PO4 out of the Tdp2 active site (Figure 4D).	RESULTS
147	153	5′-PO4	chemical	Interestingly, bi-dentate inner sphere metal contacts from the Ca2+ ion to Glu162 distort the active site phosphate-binding mode, and dislodge the 5′-PO4 out of the Tdp2 active site (Figure 4D).	RESULTS
165	169	Tdp2	protein	Interestingly, bi-dentate inner sphere metal contacts from the Ca2+ ion to Glu162 distort the active site phosphate-binding mode, and dislodge the 5′-PO4 out of the Tdp2 active site (Figure 4D).	RESULTS
170	181	active site	site	Interestingly, bi-dentate inner sphere metal contacts from the Ca2+ ion to Glu162 distort the active site phosphate-binding mode, and dislodge the 5′-PO4 out of the Tdp2 active site (Figure 4D).	RESULTS
71	75	Ca2+	chemical	Together with results showing that under the conditions examined here, Ca2+ inhibits rather than stimulates the Tdp2 reaction, the divalent metal bound Tdp2 structures provide a mechanism for Ca2+-mediated inhibition of the Tdp2 reaction.	RESULTS
112	116	Tdp2	protein	Together with results showing that under the conditions examined here, Ca2+ inhibits rather than stimulates the Tdp2 reaction, the divalent metal bound Tdp2 structures provide a mechanism for Ca2+-mediated inhibition of the Tdp2 reaction.	RESULTS
131	151	divalent metal bound	protein_state	Together with results showing that under the conditions examined here, Ca2+ inhibits rather than stimulates the Tdp2 reaction, the divalent metal bound Tdp2 structures provide a mechanism for Ca2+-mediated inhibition of the Tdp2 reaction.	RESULTS
152	156	Tdp2	protein	Together with results showing that under the conditions examined here, Ca2+ inhibits rather than stimulates the Tdp2 reaction, the divalent metal bound Tdp2 structures provide a mechanism for Ca2+-mediated inhibition of the Tdp2 reaction.	RESULTS
157	167	structures	evidence	Together with results showing that under the conditions examined here, Ca2+ inhibits rather than stimulates the Tdp2 reaction, the divalent metal bound Tdp2 structures provide a mechanism for Ca2+-mediated inhibition of the Tdp2 reaction.	RESULTS
192	196	Ca2+	chemical	Together with results showing that under the conditions examined here, Ca2+ inhibits rather than stimulates the Tdp2 reaction, the divalent metal bound Tdp2 structures provide a mechanism for Ca2+-mediated inhibition of the Tdp2 reaction.	RESULTS
224	228	Tdp2	protein	Together with results showing that under the conditions examined here, Ca2+ inhibits rather than stimulates the Tdp2 reaction, the divalent metal bound Tdp2 structures provide a mechanism for Ca2+-mediated inhibition of the Tdp2 reaction.	RESULTS
13	17	Tdp2	protein	Modeling the Tdp2 reaction coordinate	RESULTS
56	60	Mg2+	chemical	Next, to examine the feasibility of our proposed single Mg2+ mechanism, we simulated the Tdp2 reaction coordinate with hybrid QM/MM modeling using Tdp2 substrate analog- and product-bound structures as guides.	RESULTS
75	84	simulated	experimental_method	Next, to examine the feasibility of our proposed single Mg2+ mechanism, we simulated the Tdp2 reaction coordinate with hybrid QM/MM modeling using Tdp2 substrate analog- and product-bound structures as guides.	RESULTS
89	93	Tdp2	protein	Next, to examine the feasibility of our proposed single Mg2+ mechanism, we simulated the Tdp2 reaction coordinate with hybrid QM/MM modeling using Tdp2 substrate analog- and product-bound structures as guides.	RESULTS
119	140	hybrid QM/MM modeling	experimental_method	Next, to examine the feasibility of our proposed single Mg2+ mechanism, we simulated the Tdp2 reaction coordinate with hybrid QM/MM modeling using Tdp2 substrate analog- and product-bound structures as guides.	RESULTS
147	151	Tdp2	protein	Next, to examine the feasibility of our proposed single Mg2+ mechanism, we simulated the Tdp2 reaction coordinate with hybrid QM/MM modeling using Tdp2 substrate analog- and product-bound structures as guides.	RESULTS
152	169	substrate analog-	protein_state	Next, to examine the feasibility of our proposed single Mg2+ mechanism, we simulated the Tdp2 reaction coordinate with hybrid QM/MM modeling using Tdp2 substrate analog- and product-bound structures as guides.	RESULTS
174	187	product-bound	protein_state	Next, to examine the feasibility of our proposed single Mg2+ mechanism, we simulated the Tdp2 reaction coordinate with hybrid QM/MM modeling using Tdp2 substrate analog- and product-bound structures as guides.	RESULTS
188	198	structures	evidence	Next, to examine the feasibility of our proposed single Mg2+ mechanism, we simulated the Tdp2 reaction coordinate with hybrid QM/MM modeling using Tdp2 substrate analog- and product-bound structures as guides.	RESULTS
9	28	structural analyses	experimental_method	Previous structural analyses showed that the superposition of a DNA substrate mimic (5′-aminohexanol) and product (5′-PO4) complexes delineates a probable Tdp2 reaction trajectory characterized by inversion of stereochemistry about the adducted 5′-phosphorus.	RESULTS
45	58	superposition	experimental_method	Previous structural analyses showed that the superposition of a DNA substrate mimic (5′-aminohexanol) and product (5′-PO4) complexes delineates a probable Tdp2 reaction trajectory characterized by inversion of stereochemistry about the adducted 5′-phosphorus.	RESULTS
64	67	DNA	chemical	Previous structural analyses showed that the superposition of a DNA substrate mimic (5′-aminohexanol) and product (5′-PO4) complexes delineates a probable Tdp2 reaction trajectory characterized by inversion of stereochemistry about the adducted 5′-phosphorus.	RESULTS
85	100	5′-aminohexanol	chemical	Previous structural analyses showed that the superposition of a DNA substrate mimic (5′-aminohexanol) and product (5′-PO4) complexes delineates a probable Tdp2 reaction trajectory characterized by inversion of stereochemistry about the adducted 5′-phosphorus.	RESULTS
115	121	5′-PO4	chemical	Previous structural analyses showed that the superposition of a DNA substrate mimic (5′-aminohexanol) and product (5′-PO4) complexes delineates a probable Tdp2 reaction trajectory characterized by inversion of stereochemistry about the adducted 5′-phosphorus.	RESULTS
155	159	Tdp2	protein	Previous structural analyses showed that the superposition of a DNA substrate mimic (5′-aminohexanol) and product (5′-PO4) complexes delineates a probable Tdp2 reaction trajectory characterized by inversion of stereochemistry about the adducted 5′-phosphorus.	RESULTS
53	58	water	chemical	In this scheme (Figure 5A), a candidate nucleophilic water that is strongly hydrogen bonded to Asp272 and Asn274, is well positioned for the in-line nucleophilic attack ∼180° opposite of the P–O bond of the 5′-Tyr adduct.	RESULTS
76	91	hydrogen bonded	bond_interaction	In this scheme (Figure 5A), a candidate nucleophilic water that is strongly hydrogen bonded to Asp272 and Asn274, is well positioned for the in-line nucleophilic attack ∼180° opposite of the P–O bond of the 5′-Tyr adduct.	RESULTS
95	101	Asp272	residue_name_number	In this scheme (Figure 5A), a candidate nucleophilic water that is strongly hydrogen bonded to Asp272 and Asn274, is well positioned for the in-line nucleophilic attack ∼180° opposite of the P–O bond of the 5′-Tyr adduct.	RESULTS
106	112	Asn274	residue_name_number	In this scheme (Figure 5A), a candidate nucleophilic water that is strongly hydrogen bonded to Asp272 and Asn274, is well positioned for the in-line nucleophilic attack ∼180° opposite of the P–O bond of the 5′-Tyr adduct.	RESULTS
35	39	Tdp2	protein	Structure-function analysis of the Tdp2 reaction mechanism.	FIG
41	56	phosphotyrosine	residue_name	(A) Proposed mechanism for hydrolysis of phosphotyrosine bond by Tdp2.	FIG
65	69	Tdp2	protein	(A) Proposed mechanism for hydrolysis of phosphotyrosine bond by Tdp2.	FIG
27	39	binding-site	site	Residues in green form the binding-site for the 5′-tyrosine (red) and phosphate, yellow bind the 5′ nucleotide and blue bind nucleotides 2–3.	FIG
48	59	5′-tyrosine	residue_name	Residues in green form the binding-site for the 5′-tyrosine (red) and phosphate, yellow bind the 5′ nucleotide and blue bind nucleotides 2–3.	FIG
70	79	phosphate	chemical	Residues in green form the binding-site for the 5′-tyrosine (red) and phosphate, yellow bind the 5′ nucleotide and blue bind nucleotides 2–3.	FIG
34	39	mTdp2	protein	Residue numbers shown are for the mTdp2 homolog. (B) Free energy during the QM/MM simulation as a function of distance between the nucleophilic water and 5′-phosphorus atom.	FIG
53	64	Free energy	evidence	Residue numbers shown are for the mTdp2 homolog. (B) Free energy during the QM/MM simulation as a function of distance between the nucleophilic water and 5′-phosphorus atom.	FIG
76	92	QM/MM simulation	experimental_method	Residue numbers shown are for the mTdp2 homolog. (B) Free energy during the QM/MM simulation as a function of distance between the nucleophilic water and 5′-phosphorus atom.	FIG
144	149	water	chemical	Residue numbers shown are for the mTdp2 homolog. (B) Free energy during the QM/MM simulation as a function of distance between the nucleophilic water and 5′-phosphorus atom.	FIG
57	69	mTdp2cat-DNA	complex_assembly	Reaction proceeds from right to left. (C) Models for the mTdp2cat-DNA complex during the QM/MM reaction path simulation showing the substrate (left, tan), transition state intermediate (center, cyan) and product (right, pink) states.	FIG
89	119	QM/MM reaction path simulation	experimental_method	Reaction proceeds from right to left. (C) Models for the mTdp2cat-DNA complex during the QM/MM reaction path simulation showing the substrate (left, tan), transition state intermediate (center, cyan) and product (right, pink) states.	FIG
34	39	mTdp2	protein	Residue numbers shown are for the mTdp2 homolog. (D) Electrostatic surface potential calculated for 5′-phosphotyrosine in isolation (upper panel) and in the presence of a cation–π interaction with the guanidinium group of Arg216 (lower panel) shows electron-withdrawing effect of this interaction.	FIG
53	84	Electrostatic surface potential	evidence	Residue numbers shown are for the mTdp2 homolog. (D) Electrostatic surface potential calculated for 5′-phosphotyrosine in isolation (upper panel) and in the presence of a cation–π interaction with the guanidinium group of Arg216 (lower panel) shows electron-withdrawing effect of this interaction.	FIG
100	118	5′-phosphotyrosine	residue_name	Residue numbers shown are for the mTdp2 homolog. (D) Electrostatic surface potential calculated for 5′-phosphotyrosine in isolation (upper panel) and in the presence of a cation–π interaction with the guanidinium group of Arg216 (lower panel) shows electron-withdrawing effect of this interaction.	FIG
157	168	presence of	protein_state	Residue numbers shown are for the mTdp2 homolog. (D) Electrostatic surface potential calculated for 5′-phosphotyrosine in isolation (upper panel) and in the presence of a cation–π interaction with the guanidinium group of Arg216 (lower panel) shows electron-withdrawing effect of this interaction.	FIG
171	191	cation–π interaction	bond_interaction	Residue numbers shown are for the mTdp2 homolog. (D) Electrostatic surface potential calculated for 5′-phosphotyrosine in isolation (upper panel) and in the presence of a cation–π interaction with the guanidinium group of Arg216 (lower panel) shows electron-withdrawing effect of this interaction.	FIG
222	228	Arg216	residue_name_number	Residue numbers shown are for the mTdp2 homolog. (D) Electrostatic surface potential calculated for 5′-phosphotyrosine in isolation (upper panel) and in the presence of a cation–π interaction with the guanidinium group of Arg216 (lower panel) shows electron-withdrawing effect of this interaction.	FIG
0	23	Electrostatic potential	evidence	Electrostatic potential color gradient extends from positive (red) through neutral (gray), to negative (blue). (E) Bar graph displaying the relative activity of wild-type and mutant human MBP-hTdp2cat fusion proteins on the three substrates.	FIG
161	170	wild-type	protein_state	Electrostatic potential color gradient extends from positive (red) through neutral (gray), to negative (blue). (E) Bar graph displaying the relative activity of wild-type and mutant human MBP-hTdp2cat fusion proteins on the three substrates.	FIG
175	181	mutant	protein_state	Electrostatic potential color gradient extends from positive (red) through neutral (gray), to negative (blue). (E) Bar graph displaying the relative activity of wild-type and mutant human MBP-hTdp2cat fusion proteins on the three substrates.	FIG
182	187	human	species	Electrostatic potential color gradient extends from positive (red) through neutral (gray), to negative (blue). (E) Bar graph displaying the relative activity of wild-type and mutant human MBP-hTdp2cat fusion proteins on the three substrates.	FIG
188	191	MBP	experimental_method	Electrostatic potential color gradient extends from positive (red) through neutral (gray), to negative (blue). (E) Bar graph displaying the relative activity of wild-type and mutant human MBP-hTdp2cat fusion proteins on the three substrates.	FIG
192	200	hTdp2cat	structure_element	Electrostatic potential color gradient extends from positive (red) through neutral (gray), to negative (blue). (E) Bar graph displaying the relative activity of wild-type and mutant human MBP-hTdp2cat fusion proteins on the three substrates.	FIG
201	216	fusion proteins	experimental_method	Electrostatic potential color gradient extends from positive (red) through neutral (gray), to negative (blue). (E) Bar graph displaying the relative activity of wild-type and mutant human MBP-hTdp2cat fusion proteins on the three substrates.	FIG
11	14	PNP	chemical	Release of PNP from PNP phosphate and T5PNP was detected as an increase in absorbance at 415 nm.	FIG
20	23	PNP	chemical	Release of PNP from PNP phosphate and T5PNP was detected as an increase in absorbance at 415 nm.	FIG
24	33	phosphate	chemical	Release of PNP from PNP phosphate and T5PNP was detected as an increase in absorbance at 415 nm.	FIG
38	43	T5PNP	chemical	Release of PNP from PNP phosphate and T5PNP was detected as an increase in absorbance at 415 nm.	FIG
0	14	Reaction rates	evidence	Reaction rates are expressed as the percent of activity relative to wildtype MBP-hTdp2cat; error bars, s.d.	FIG
68	76	wildtype	protein_state	Reaction rates are expressed as the percent of activity relative to wildtype MBP-hTdp2cat; error bars, s.d.	FIG
77	80	MBP	experimental_method	Reaction rates are expressed as the percent of activity relative to wildtype MBP-hTdp2cat; error bars, s.d.	FIG
81	89	hTdp2cat	structure_element	Reaction rates are expressed as the percent of activity relative to wildtype MBP-hTdp2cat; error bars, s.d.	FIG
11	16	hTdp2	protein	Mutants of hTdp2 (black) and the equivalent residue in mTdp2 (tan) are indicated.	FIG
55	60	mTdp2	protein	Mutants of hTdp2 (black) and the equivalent residue in mTdp2 (tan) are indicated.	FIG
65	70	water	chemical	We examined the energy profile of the nucleophilic attack of the water molecule by using the distance between the water oxygen and the P atom on the phosphate moiety as the sole reaction coordinate in the present calculation (Figure 5B and C).	RESULTS
114	119	water	chemical	We examined the energy profile of the nucleophilic attack of the water molecule by using the distance between the water oxygen and the P atom on the phosphate moiety as the sole reaction coordinate in the present calculation (Figure 5B and C).	RESULTS
62	70	mTdp2cat	structure_element	A starting model was generated from atomic coordinates of the mTdp2cat 5′–aminohexanol substrate analog structure (PDB 4GZ0) with a tyrosine replacing the 5′-aminohexanol then adding the Mg2+ and inner-sphere waters from the mTdp2-DNA product structure (PDB, 4GZ1), and running an initial round of molecular dynamics simulation (10 ns) to allow the system to reach an equilibrium.	RESULTS
71	86	5′–aminohexanol	chemical	A starting model was generated from atomic coordinates of the mTdp2cat 5′–aminohexanol substrate analog structure (PDB 4GZ0) with a tyrosine replacing the 5′-aminohexanol then adding the Mg2+ and inner-sphere waters from the mTdp2-DNA product structure (PDB, 4GZ1), and running an initial round of molecular dynamics simulation (10 ns) to allow the system to reach an equilibrium.	RESULTS
104	113	structure	evidence	A starting model was generated from atomic coordinates of the mTdp2cat 5′–aminohexanol substrate analog structure (PDB 4GZ0) with a tyrosine replacing the 5′-aminohexanol then adding the Mg2+ and inner-sphere waters from the mTdp2-DNA product structure (PDB, 4GZ1), and running an initial round of molecular dynamics simulation (10 ns) to allow the system to reach an equilibrium.	RESULTS
132	140	tyrosine	residue_name	A starting model was generated from atomic coordinates of the mTdp2cat 5′–aminohexanol substrate analog structure (PDB 4GZ0) with a tyrosine replacing the 5′-aminohexanol then adding the Mg2+ and inner-sphere waters from the mTdp2-DNA product structure (PDB, 4GZ1), and running an initial round of molecular dynamics simulation (10 ns) to allow the system to reach an equilibrium.	RESULTS
155	170	5′-aminohexanol	chemical	A starting model was generated from atomic coordinates of the mTdp2cat 5′–aminohexanol substrate analog structure (PDB 4GZ0) with a tyrosine replacing the 5′-aminohexanol then adding the Mg2+ and inner-sphere waters from the mTdp2-DNA product structure (PDB, 4GZ1), and running an initial round of molecular dynamics simulation (10 ns) to allow the system to reach an equilibrium.	RESULTS
187	191	Mg2+	chemical	A starting model was generated from atomic coordinates of the mTdp2cat 5′–aminohexanol substrate analog structure (PDB 4GZ0) with a tyrosine replacing the 5′-aminohexanol then adding the Mg2+ and inner-sphere waters from the mTdp2-DNA product structure (PDB, 4GZ1), and running an initial round of molecular dynamics simulation (10 ns) to allow the system to reach an equilibrium.	RESULTS
209	215	waters	chemical	A starting model was generated from atomic coordinates of the mTdp2cat 5′–aminohexanol substrate analog structure (PDB 4GZ0) with a tyrosine replacing the 5′-aminohexanol then adding the Mg2+ and inner-sphere waters from the mTdp2-DNA product structure (PDB, 4GZ1), and running an initial round of molecular dynamics simulation (10 ns) to allow the system to reach an equilibrium.	RESULTS
225	234	mTdp2-DNA	complex_assembly	A starting model was generated from atomic coordinates of the mTdp2cat 5′–aminohexanol substrate analog structure (PDB 4GZ0) with a tyrosine replacing the 5′-aminohexanol then adding the Mg2+ and inner-sphere waters from the mTdp2-DNA product structure (PDB, 4GZ1), and running an initial round of molecular dynamics simulation (10 ns) to allow the system to reach an equilibrium.	RESULTS
243	252	structure	evidence	A starting model was generated from atomic coordinates of the mTdp2cat 5′–aminohexanol substrate analog structure (PDB 4GZ0) with a tyrosine replacing the 5′-aminohexanol then adding the Mg2+ and inner-sphere waters from the mTdp2-DNA product structure (PDB, 4GZ1), and running an initial round of molecular dynamics simulation (10 ns) to allow the system to reach an equilibrium.	RESULTS
298	327	molecular dynamics simulation	experimental_method	A starting model was generated from atomic coordinates of the mTdp2cat 5′–aminohexanol substrate analog structure (PDB 4GZ0) with a tyrosine replacing the 5′-aminohexanol then adding the Mg2+ and inner-sphere waters from the mTdp2-DNA product structure (PDB, 4GZ1), and running an initial round of molecular dynamics simulation (10 ns) to allow the system to reach an equilibrium.	RESULTS
6	24	QM/MM optimization	experimental_method	After QM/MM optimization of this model (Figure 5C, ‘i-substrate’), the O–P distance is 3.4 Å, which is in agreement with the range of distances observed in the mTdp2cat 5′-aminohexanol substrate analog structure (3.2–3.4 Å).	RESULTS
160	168	mTdp2cat	structure_element	After QM/MM optimization of this model (Figure 5C, ‘i-substrate’), the O–P distance is 3.4 Å, which is in agreement with the range of distances observed in the mTdp2cat 5′-aminohexanol substrate analog structure (3.2–3.4 Å).	RESULTS
169	184	5′-aminohexanol	chemical	After QM/MM optimization of this model (Figure 5C, ‘i-substrate’), the O–P distance is 3.4 Å, which is in agreement with the range of distances observed in the mTdp2cat 5′-aminohexanol substrate analog structure (3.2–3.4 Å).	RESULTS
202	211	structure	evidence	After QM/MM optimization of this model (Figure 5C, ‘i-substrate’), the O–P distance is 3.4 Å, which is in agreement with the range of distances observed in the mTdp2cat 5′-aminohexanol substrate analog structure (3.2–3.4 Å).	RESULTS
10	15	water	chemical	Here, the water proton and the neighboring O of Asp272 participates in a strong hydrogen bond (distance of 1.58 Å) and the phosphotyrosyl O–P distance is stretched to 1.77 Å, which is 0.1 Å beyond an equilibrium bond length.	RESULTS
48	54	Asp272	residue_name_number	Here, the water proton and the neighboring O of Asp272 participates in a strong hydrogen bond (distance of 1.58 Å) and the phosphotyrosyl O–P distance is stretched to 1.77 Å, which is 0.1 Å beyond an equilibrium bond length.	RESULTS
80	93	hydrogen bond	bond_interaction	Here, the water proton and the neighboring O of Asp272 participates in a strong hydrogen bond (distance of 1.58 Å) and the phosphotyrosyl O–P distance is stretched to 1.77 Å, which is 0.1 Å beyond an equilibrium bond length.	RESULTS
123	137	phosphotyrosyl	ptm	Here, the water proton and the neighboring O of Asp272 participates in a strong hydrogen bond (distance of 1.58 Å) and the phosphotyrosyl O–P distance is stretched to 1.77 Å, which is 0.1 Å beyond an equilibrium bond length.	RESULTS
35	45	simulation	experimental_method	In the subsequent two steps of the simulation, as the water-phosphate O–P distance reduces to 1.98 Å, a key hydrogen bond between the nucleophilic water and Asp272 shortens to 1.38 Å as the water H–O bond approaches the point of dissociation.	RESULTS
54	59	water	chemical	In the subsequent two steps of the simulation, as the water-phosphate O–P distance reduces to 1.98 Å, a key hydrogen bond between the nucleophilic water and Asp272 shortens to 1.38 Å as the water H–O bond approaches the point of dissociation.	RESULTS
108	121	hydrogen bond	bond_interaction	In the subsequent two steps of the simulation, as the water-phosphate O–P distance reduces to 1.98 Å, a key hydrogen bond between the nucleophilic water and Asp272 shortens to 1.38 Å as the water H–O bond approaches the point of dissociation.	RESULTS
147	152	water	chemical	In the subsequent two steps of the simulation, as the water-phosphate O–P distance reduces to 1.98 Å, a key hydrogen bond between the nucleophilic water and Asp272 shortens to 1.38 Å as the water H–O bond approaches the point of dissociation.	RESULTS
157	163	Asp272	residue_name_number	In the subsequent two steps of the simulation, as the water-phosphate O–P distance reduces to 1.98 Å, a key hydrogen bond between the nucleophilic water and Asp272 shortens to 1.38 Å as the water H–O bond approaches the point of dissociation.	RESULTS
190	195	water	chemical	In the subsequent two steps of the simulation, as the water-phosphate O–P distance reduces to 1.98 Å, a key hydrogen bond between the nucleophilic water and Asp272 shortens to 1.38 Å as the water H–O bond approaches the point of dissociation.	RESULTS
25	30	water	chemical	The second proton on the water nucleophile maintains a strong hydrogen bond with Asn274 throughout the reaction, implicating this residue in orienting the water nucleophile during the reaction.	RESULTS
62	75	hydrogen bond	bond_interaction	The second proton on the water nucleophile maintains a strong hydrogen bond with Asn274 throughout the reaction, implicating this residue in orienting the water nucleophile during the reaction.	RESULTS
81	87	Asn274	residue_name_number	The second proton on the water nucleophile maintains a strong hydrogen bond with Asn274 throughout the reaction, implicating this residue in orienting the water nucleophile during the reaction.	RESULTS
155	160	water	chemical	The second proton on the water nucleophile maintains a strong hydrogen bond with Asn274 throughout the reaction, implicating this residue in orienting the water nucleophile during the reaction.	RESULTS
27	41	phosphotyrosyl	ptm	Concomitant with this, the phosphotyrosyl O–P bond weakens (d = 1.89 Å), and the formation of the penta-covalent transition state (Figure 5C ‘ii-transition state’) is observed.	RESULTS
57	66	phosphate	chemical	The final steps show inversion of stereochemistry at the phosphate, along with lengthening and breaking of the phosphotyrosyl O–P bond.	RESULTS
111	125	phosphotyrosyl	ptm	The final steps show inversion of stereochemistry at the phosphate, along with lengthening and breaking of the phosphotyrosyl O–P bond.	RESULTS
78	83	water	chemical	Product formation is coupled to a transfer of a proton from the nucleophillic water to Asp272, consistent with the proposed function for this residue as the catalytic base.	RESULTS
87	93	Asp272	residue_name_number	Product formation is coupled to a transfer of a proton from the nucleophillic water to Asp272, consistent with the proposed function for this residue as the catalytic base.	RESULTS
55	62	His 359	residue_name_number	Of note, both nitrogens of the imidazole side chain of His 359 require protonation for stability of the simulation.	RESULTS
104	114	simulation	experimental_method	Of note, both nitrogens of the imidazole side chain of His 359 require protonation for stability of the simulation.	RESULTS
0	7	Asp 326	residue_name_number	Asp 326 makes a hydrogen bond to N∂1 of His359, suggesting that this salt bridge could stabilize the protonated form of His359 as has been demonstrated for the analogous Asp-His pair in the EEP domain of APE1, which elevates the pKa of this His above 8.0. In our model, the transition state contains a hydrogen bond between the doubly protonated His359 and the phosphate oxygen that also coordinates with the single catalytic Mg2+, while the second His359 imidazole proton maintains a H-bond with the Asp326 residue throughout the reaction.	RESULTS
16	29	hydrogen bond	bond_interaction	Asp 326 makes a hydrogen bond to N∂1 of His359, suggesting that this salt bridge could stabilize the protonated form of His359 as has been demonstrated for the analogous Asp-His pair in the EEP domain of APE1, which elevates the pKa of this His above 8.0. In our model, the transition state contains a hydrogen bond between the doubly protonated His359 and the phosphate oxygen that also coordinates with the single catalytic Mg2+, while the second His359 imidazole proton maintains a H-bond with the Asp326 residue throughout the reaction.	RESULTS
40	46	His359	residue_name_number	Asp 326 makes a hydrogen bond to N∂1 of His359, suggesting that this salt bridge could stabilize the protonated form of His359 as has been demonstrated for the analogous Asp-His pair in the EEP domain of APE1, which elevates the pKa of this His above 8.0. In our model, the transition state contains a hydrogen bond between the doubly protonated His359 and the phosphate oxygen that also coordinates with the single catalytic Mg2+, while the second His359 imidazole proton maintains a H-bond with the Asp326 residue throughout the reaction.	RESULTS
69	80	salt bridge	bond_interaction	Asp 326 makes a hydrogen bond to N∂1 of His359, suggesting that this salt bridge could stabilize the protonated form of His359 as has been demonstrated for the analogous Asp-His pair in the EEP domain of APE1, which elevates the pKa of this His above 8.0. In our model, the transition state contains a hydrogen bond between the doubly protonated His359 and the phosphate oxygen that also coordinates with the single catalytic Mg2+, while the second His359 imidazole proton maintains a H-bond with the Asp326 residue throughout the reaction.	RESULTS
101	111	protonated	protein_state	Asp 326 makes a hydrogen bond to N∂1 of His359, suggesting that this salt bridge could stabilize the protonated form of His359 as has been demonstrated for the analogous Asp-His pair in the EEP domain of APE1, which elevates the pKa of this His above 8.0. In our model, the transition state contains a hydrogen bond between the doubly protonated His359 and the phosphate oxygen that also coordinates with the single catalytic Mg2+, while the second His359 imidazole proton maintains a H-bond with the Asp326 residue throughout the reaction.	RESULTS
120	126	His359	residue_name_number	Asp 326 makes a hydrogen bond to N∂1 of His359, suggesting that this salt bridge could stabilize the protonated form of His359 as has been demonstrated for the analogous Asp-His pair in the EEP domain of APE1, which elevates the pKa of this His above 8.0. In our model, the transition state contains a hydrogen bond between the doubly protonated His359 and the phosphate oxygen that also coordinates with the single catalytic Mg2+, while the second His359 imidazole proton maintains a H-bond with the Asp326 residue throughout the reaction.	RESULTS
170	173	Asp	residue_name	Asp 326 makes a hydrogen bond to N∂1 of His359, suggesting that this salt bridge could stabilize the protonated form of His359 as has been demonstrated for the analogous Asp-His pair in the EEP domain of APE1, which elevates the pKa of this His above 8.0. In our model, the transition state contains a hydrogen bond between the doubly protonated His359 and the phosphate oxygen that also coordinates with the single catalytic Mg2+, while the second His359 imidazole proton maintains a H-bond with the Asp326 residue throughout the reaction.	RESULTS
174	177	His	residue_name	Asp 326 makes a hydrogen bond to N∂1 of His359, suggesting that this salt bridge could stabilize the protonated form of His359 as has been demonstrated for the analogous Asp-His pair in the EEP domain of APE1, which elevates the pKa of this His above 8.0. In our model, the transition state contains a hydrogen bond between the doubly protonated His359 and the phosphate oxygen that also coordinates with the single catalytic Mg2+, while the second His359 imidazole proton maintains a H-bond with the Asp326 residue throughout the reaction.	RESULTS
190	193	EEP	structure_element	Asp 326 makes a hydrogen bond to N∂1 of His359, suggesting that this salt bridge could stabilize the protonated form of His359 as has been demonstrated for the analogous Asp-His pair in the EEP domain of APE1, which elevates the pKa of this His above 8.0. In our model, the transition state contains a hydrogen bond between the doubly protonated His359 and the phosphate oxygen that also coordinates with the single catalytic Mg2+, while the second His359 imidazole proton maintains a H-bond with the Asp326 residue throughout the reaction.	RESULTS
204	208	APE1	protein	Asp 326 makes a hydrogen bond to N∂1 of His359, suggesting that this salt bridge could stabilize the protonated form of His359 as has been demonstrated for the analogous Asp-His pair in the EEP domain of APE1, which elevates the pKa of this His above 8.0. In our model, the transition state contains a hydrogen bond between the doubly protonated His359 and the phosphate oxygen that also coordinates with the single catalytic Mg2+, while the second His359 imidazole proton maintains a H-bond with the Asp326 residue throughout the reaction.	RESULTS
229	232	pKa	evidence	Asp 326 makes a hydrogen bond to N∂1 of His359, suggesting that this salt bridge could stabilize the protonated form of His359 as has been demonstrated for the analogous Asp-His pair in the EEP domain of APE1, which elevates the pKa of this His above 8.0. In our model, the transition state contains a hydrogen bond between the doubly protonated His359 and the phosphate oxygen that also coordinates with the single catalytic Mg2+, while the second His359 imidazole proton maintains a H-bond with the Asp326 residue throughout the reaction.	RESULTS
241	244	His	residue_name	Asp 326 makes a hydrogen bond to N∂1 of His359, suggesting that this salt bridge could stabilize the protonated form of His359 as has been demonstrated for the analogous Asp-His pair in the EEP domain of APE1, which elevates the pKa of this His above 8.0. In our model, the transition state contains a hydrogen bond between the doubly protonated His359 and the phosphate oxygen that also coordinates with the single catalytic Mg2+, while the second His359 imidazole proton maintains a H-bond with the Asp326 residue throughout the reaction.	RESULTS
302	315	hydrogen bond	bond_interaction	Asp 326 makes a hydrogen bond to N∂1 of His359, suggesting that this salt bridge could stabilize the protonated form of His359 as has been demonstrated for the analogous Asp-His pair in the EEP domain of APE1, which elevates the pKa of this His above 8.0. In our model, the transition state contains a hydrogen bond between the doubly protonated His359 and the phosphate oxygen that also coordinates with the single catalytic Mg2+, while the second His359 imidazole proton maintains a H-bond with the Asp326 residue throughout the reaction.	RESULTS
328	345	doubly protonated	protein_state	Asp 326 makes a hydrogen bond to N∂1 of His359, suggesting that this salt bridge could stabilize the protonated form of His359 as has been demonstrated for the analogous Asp-His pair in the EEP domain of APE1, which elevates the pKa of this His above 8.0. In our model, the transition state contains a hydrogen bond between the doubly protonated His359 and the phosphate oxygen that also coordinates with the single catalytic Mg2+, while the second His359 imidazole proton maintains a H-bond with the Asp326 residue throughout the reaction.	RESULTS
346	352	His359	residue_name_number	Asp 326 makes a hydrogen bond to N∂1 of His359, suggesting that this salt bridge could stabilize the protonated form of His359 as has been demonstrated for the analogous Asp-His pair in the EEP domain of APE1, which elevates the pKa of this His above 8.0. In our model, the transition state contains a hydrogen bond between the doubly protonated His359 and the phosphate oxygen that also coordinates with the single catalytic Mg2+, while the second His359 imidazole proton maintains a H-bond with the Asp326 residue throughout the reaction.	RESULTS
361	370	phosphate	chemical	Asp 326 makes a hydrogen bond to N∂1 of His359, suggesting that this salt bridge could stabilize the protonated form of His359 as has been demonstrated for the analogous Asp-His pair in the EEP domain of APE1, which elevates the pKa of this His above 8.0. In our model, the transition state contains a hydrogen bond between the doubly protonated His359 and the phosphate oxygen that also coordinates with the single catalytic Mg2+, while the second His359 imidazole proton maintains a H-bond with the Asp326 residue throughout the reaction.	RESULTS
426	430	Mg2+	chemical	Asp 326 makes a hydrogen bond to N∂1 of His359, suggesting that this salt bridge could stabilize the protonated form of His359 as has been demonstrated for the analogous Asp-His pair in the EEP domain of APE1, which elevates the pKa of this His above 8.0. In our model, the transition state contains a hydrogen bond between the doubly protonated His359 and the phosphate oxygen that also coordinates with the single catalytic Mg2+, while the second His359 imidazole proton maintains a H-bond with the Asp326 residue throughout the reaction.	RESULTS
449	455	His359	residue_name_number	Asp 326 makes a hydrogen bond to N∂1 of His359, suggesting that this salt bridge could stabilize the protonated form of His359 as has been demonstrated for the analogous Asp-His pair in the EEP domain of APE1, which elevates the pKa of this His above 8.0. In our model, the transition state contains a hydrogen bond between the doubly protonated His359 and the phosphate oxygen that also coordinates with the single catalytic Mg2+, while the second His359 imidazole proton maintains a H-bond with the Asp326 residue throughout the reaction.	RESULTS
485	491	H-bond	bond_interaction	Asp 326 makes a hydrogen bond to N∂1 of His359, suggesting that this salt bridge could stabilize the protonated form of His359 as has been demonstrated for the analogous Asp-His pair in the EEP domain of APE1, which elevates the pKa of this His above 8.0. In our model, the transition state contains a hydrogen bond between the doubly protonated His359 and the phosphate oxygen that also coordinates with the single catalytic Mg2+, while the second His359 imidazole proton maintains a H-bond with the Asp326 residue throughout the reaction.	RESULTS
501	507	Asp326	residue_name_number	Asp 326 makes a hydrogen bond to N∂1 of His359, suggesting that this salt bridge could stabilize the protonated form of His359 as has been demonstrated for the analogous Asp-His pair in the EEP domain of APE1, which elevates the pKa of this His above 8.0. In our model, the transition state contains a hydrogen bond between the doubly protonated His359 and the phosphate oxygen that also coordinates with the single catalytic Mg2+, while the second His359 imidazole proton maintains a H-bond with the Asp326 residue throughout the reaction.	RESULTS
23	32	structure	evidence	In the final optimized structure, the observed product state (Figure 5C, ‘iii-product’) is found in a conformation that is 7.4 kcal mol−1 more stable than the initial reactive state (Figure 5B).	RESULTS
4	12	tyrosine	residue_name	The tyrosine oxy-anion product is coordinated to the Mg2+ ion with a 2.0 Å distance, which is the shortest of the six Mg2+ ligands (including three water molecules, one of the free oxygens on the phosphate group and the Glu162 residue), indicating the single Mg2+ greatly stabilizes the product oxy-anion.	RESULTS
34	48	coordinated to	bond_interaction	The tyrosine oxy-anion product is coordinated to the Mg2+ ion with a 2.0 Å distance, which is the shortest of the six Mg2+ ligands (including three water molecules, one of the free oxygens on the phosphate group and the Glu162 residue), indicating the single Mg2+ greatly stabilizes the product oxy-anion.	RESULTS
53	57	Mg2+	chemical	The tyrosine oxy-anion product is coordinated to the Mg2+ ion with a 2.0 Å distance, which is the shortest of the six Mg2+ ligands (including three water molecules, one of the free oxygens on the phosphate group and the Glu162 residue), indicating the single Mg2+ greatly stabilizes the product oxy-anion.	RESULTS
118	122	Mg2+	chemical	The tyrosine oxy-anion product is coordinated to the Mg2+ ion with a 2.0 Å distance, which is the shortest of the six Mg2+ ligands (including three water molecules, one of the free oxygens on the phosphate group and the Glu162 residue), indicating the single Mg2+ greatly stabilizes the product oxy-anion.	RESULTS
148	153	water	chemical	The tyrosine oxy-anion product is coordinated to the Mg2+ ion with a 2.0 Å distance, which is the shortest of the six Mg2+ ligands (including three water molecules, one of the free oxygens on the phosphate group and the Glu162 residue), indicating the single Mg2+ greatly stabilizes the product oxy-anion.	RESULTS
196	205	phosphate	chemical	The tyrosine oxy-anion product is coordinated to the Mg2+ ion with a 2.0 Å distance, which is the shortest of the six Mg2+ ligands (including three water molecules, one of the free oxygens on the phosphate group and the Glu162 residue), indicating the single Mg2+ greatly stabilizes the product oxy-anion.	RESULTS
220	226	Glu162	residue_name_number	The tyrosine oxy-anion product is coordinated to the Mg2+ ion with a 2.0 Å distance, which is the shortest of the six Mg2+ ligands (including three water molecules, one of the free oxygens on the phosphate group and the Glu162 residue), indicating the single Mg2+ greatly stabilizes the product oxy-anion.	RESULTS
259	263	Mg2+	chemical	The tyrosine oxy-anion product is coordinated to the Mg2+ ion with a 2.0 Å distance, which is the shortest of the six Mg2+ ligands (including three water molecules, one of the free oxygens on the phosphate group and the Glu162 residue), indicating the single Mg2+ greatly stabilizes the product oxy-anion.	RESULTS
48	62	QM/MM modeling	experimental_method	An additional striking feature gleaned from the QM/MM modeling is the putative binding mode of the Top2 tyrosine-leaving group.	RESULTS
99	103	Top2	protein_type	An additional striking feature gleaned from the QM/MM modeling is the putative binding mode of the Top2 tyrosine-leaving group.	RESULTS
104	112	tyrosine	residue_name	An additional striking feature gleaned from the QM/MM modeling is the putative binding mode of the Top2 tyrosine-leaving group.	RESULTS
10	19	conserved	protein_state	A trio of conserved residues (Tyr 188, Arg 216 and Ser 239) forms the walls of a conserved Top2 tyrosine binding pocket.	RESULTS
30	37	Tyr 188	residue_name_number	A trio of conserved residues (Tyr 188, Arg 216 and Ser 239) forms the walls of a conserved Top2 tyrosine binding pocket.	RESULTS
39	46	Arg 216	residue_name_number	A trio of conserved residues (Tyr 188, Arg 216 and Ser 239) forms the walls of a conserved Top2 tyrosine binding pocket.	RESULTS
51	58	Ser 239	residue_name_number	A trio of conserved residues (Tyr 188, Arg 216 and Ser 239) forms the walls of a conserved Top2 tyrosine binding pocket.	RESULTS
81	90	conserved	protein_state	A trio of conserved residues (Tyr 188, Arg 216 and Ser 239) forms the walls of a conserved Top2 tyrosine binding pocket.	RESULTS
91	95	Top2	protein_type	A trio of conserved residues (Tyr 188, Arg 216 and Ser 239) forms the walls of a conserved Top2 tyrosine binding pocket.	RESULTS
96	119	tyrosine binding pocket	site	A trio of conserved residues (Tyr 188, Arg 216 and Ser 239) forms the walls of a conserved Top2 tyrosine binding pocket.	RESULTS
16	36	cation–π interaction	bond_interaction	We propose this cation–π interaction further contributes to tuned stabilization of the negatively charged phenolate reaction product.	RESULTS
34	57	electrostatic potential	evidence	Consistent with this, analysis of electrostatic potential of the phosphotyrosyl moiety using Gaussian 09.D01 in the presence and absence of the Arg216 guanidinium reveals Arg216 is strongly electron withdrawing (Figure 5D).	RESULTS
65	79	phosphotyrosyl	ptm	Consistent with this, analysis of electrostatic potential of the phosphotyrosyl moiety using Gaussian 09.D01 in the presence and absence of the Arg216 guanidinium reveals Arg216 is strongly electron withdrawing (Figure 5D).	RESULTS
116	124	presence	protein_state	Consistent with this, analysis of electrostatic potential of the phosphotyrosyl moiety using Gaussian 09.D01 in the presence and absence of the Arg216 guanidinium reveals Arg216 is strongly electron withdrawing (Figure 5D).	RESULTS
129	139	absence of	protein_state	Consistent with this, analysis of electrostatic potential of the phosphotyrosyl moiety using Gaussian 09.D01 in the presence and absence of the Arg216 guanidinium reveals Arg216 is strongly electron withdrawing (Figure 5D).	RESULTS
144	150	Arg216	residue_name_number	Consistent with this, analysis of electrostatic potential of the phosphotyrosyl moiety using Gaussian 09.D01 in the presence and absence of the Arg216 guanidinium reveals Arg216 is strongly electron withdrawing (Figure 5D).	RESULTS
171	177	Arg216	residue_name_number	Consistent with this, analysis of electrostatic potential of the phosphotyrosyl moiety using Gaussian 09.D01 in the presence and absence of the Arg216 guanidinium reveals Arg216 is strongly electron withdrawing (Figure 5D).	RESULTS
45	65	cation–π interaction	bond_interaction	We further examined the contribution of this cation–π interaction to the reaction chemistry by moving the guanidinium group of Arg216 from the QM system to the MM system as either a +1 or ∼0 charge species, and re-computed energy penalties for each step in the reaction coordinate (Supplementary Figure S5A).	RESULTS
127	133	Arg216	residue_name_number	We further examined the contribution of this cation–π interaction to the reaction chemistry by moving the guanidinium group of Arg216 from the QM system to the MM system as either a +1 or ∼0 charge species, and re-computed energy penalties for each step in the reaction coordinate (Supplementary Figure S5A).	RESULTS
143	145	QM	experimental_method	We further examined the contribution of this cation–π interaction to the reaction chemistry by moving the guanidinium group of Arg216 from the QM system to the MM system as either a +1 or ∼0 charge species, and re-computed energy penalties for each step in the reaction coordinate (Supplementary Figure S5A).	RESULTS
160	162	MM	experimental_method	We further examined the contribution of this cation–π interaction to the reaction chemistry by moving the guanidinium group of Arg216 from the QM system to the MM system as either a +1 or ∼0 charge species, and re-computed energy penalties for each step in the reaction coordinate (Supplementary Figure S5A).	RESULTS
223	239	energy penalties	evidence	We further examined the contribution of this cation–π interaction to the reaction chemistry by moving the guanidinium group of Arg216 from the QM system to the MM system as either a +1 or ∼0 charge species, and re-computed energy penalties for each step in the reaction coordinate (Supplementary Figure S5A).	RESULTS
9	15	Arg216	residue_name_number	Removing Arg216 from the quantum subsystem incurs an ∼2 kcal mol−1 penalty in the transition state and product complex.	RESULTS
30	36	Arg216	residue_name_number	Removing the +1 charge on the Arg216 has a minimal impact on the transition state, but incurs an additional ∼2 kcal mol−1 penalty in the product complex.	RESULTS
12	26	QM/MM modeling	experimental_method	Altogether, QM/MM modeling identifies new determinants of the Tdp2 reaction, and demonstrates our proposed single Mg2+ catalyzed reaction model is a viable mechanism for Tdp2-catalyzed 5′-phosphotyrosine bond hydrolysis.	RESULTS
62	66	Tdp2	protein	Altogether, QM/MM modeling identifies new determinants of the Tdp2 reaction, and demonstrates our proposed single Mg2+ catalyzed reaction model is a viable mechanism for Tdp2-catalyzed 5′-phosphotyrosine bond hydrolysis.	RESULTS
114	118	Mg2+	chemical	Altogether, QM/MM modeling identifies new determinants of the Tdp2 reaction, and demonstrates our proposed single Mg2+ catalyzed reaction model is a viable mechanism for Tdp2-catalyzed 5′-phosphotyrosine bond hydrolysis.	RESULTS
170	174	Tdp2	protein	Altogether, QM/MM modeling identifies new determinants of the Tdp2 reaction, and demonstrates our proposed single Mg2+ catalyzed reaction model is a viable mechanism for Tdp2-catalyzed 5′-phosphotyrosine bond hydrolysis.	RESULTS
185	203	5′-phosphotyrosine	residue_name	Altogether, QM/MM modeling identifies new determinants of the Tdp2 reaction, and demonstrates our proposed single Mg2+ catalyzed reaction model is a viable mechanism for Tdp2-catalyzed 5′-phosphotyrosine bond hydrolysis.	RESULTS
0	4	Tdp2	protein	Tdp2 mutational analysis	RESULTS
5	24	mutational analysis	experimental_method	Tdp2 mutational analysis	RESULTS
27	31	Tdp2	protein	To test the aspects of the Tdp2 reaction mechanism described here derived from high-resolution mouse Tdp2 crystal structures (denoted with superscript numbering ‘m’ for numbering of the mouse protein), we engineered and purified thirteen human MBP-hTdp2cat mutant proteins (denoted with superscript numbering and ‘h’ for the human protein) and assayed the impacts of mutations on Tdp2 catalytic activity using three in vitro reporter substrates including a tyrosylated DNA substrate (5′-Y), p-nitrophenyl phosphate (PNPP) and thymidine 5′-monophosphate p-nitrophenyl ester (T5PNP) (Figure 5E, Supplementary Figures S5B and S5C).	RESULTS
95	100	mouse	taxonomy_domain	To test the aspects of the Tdp2 reaction mechanism described here derived from high-resolution mouse Tdp2 crystal structures (denoted with superscript numbering ‘m’ for numbering of the mouse protein), we engineered and purified thirteen human MBP-hTdp2cat mutant proteins (denoted with superscript numbering and ‘h’ for the human protein) and assayed the impacts of mutations on Tdp2 catalytic activity using three in vitro reporter substrates including a tyrosylated DNA substrate (5′-Y), p-nitrophenyl phosphate (PNPP) and thymidine 5′-monophosphate p-nitrophenyl ester (T5PNP) (Figure 5E, Supplementary Figures S5B and S5C).	RESULTS
101	105	Tdp2	protein	To test the aspects of the Tdp2 reaction mechanism described here derived from high-resolution mouse Tdp2 crystal structures (denoted with superscript numbering ‘m’ for numbering of the mouse protein), we engineered and purified thirteen human MBP-hTdp2cat mutant proteins (denoted with superscript numbering and ‘h’ for the human protein) and assayed the impacts of mutations on Tdp2 catalytic activity using three in vitro reporter substrates including a tyrosylated DNA substrate (5′-Y), p-nitrophenyl phosphate (PNPP) and thymidine 5′-monophosphate p-nitrophenyl ester (T5PNP) (Figure 5E, Supplementary Figures S5B and S5C).	RESULTS
106	124	crystal structures	evidence	To test the aspects of the Tdp2 reaction mechanism described here derived from high-resolution mouse Tdp2 crystal structures (denoted with superscript numbering ‘m’ for numbering of the mouse protein), we engineered and purified thirteen human MBP-hTdp2cat mutant proteins (denoted with superscript numbering and ‘h’ for the human protein) and assayed the impacts of mutations on Tdp2 catalytic activity using three in vitro reporter substrates including a tyrosylated DNA substrate (5′-Y), p-nitrophenyl phosphate (PNPP) and thymidine 5′-monophosphate p-nitrophenyl ester (T5PNP) (Figure 5E, Supplementary Figures S5B and S5C).	RESULTS
186	191	mouse	taxonomy_domain	To test the aspects of the Tdp2 reaction mechanism described here derived from high-resolution mouse Tdp2 crystal structures (denoted with superscript numbering ‘m’ for numbering of the mouse protein), we engineered and purified thirteen human MBP-hTdp2cat mutant proteins (denoted with superscript numbering and ‘h’ for the human protein) and assayed the impacts of mutations on Tdp2 catalytic activity using three in vitro reporter substrates including a tyrosylated DNA substrate (5′-Y), p-nitrophenyl phosphate (PNPP) and thymidine 5′-monophosphate p-nitrophenyl ester (T5PNP) (Figure 5E, Supplementary Figures S5B and S5C).	RESULTS
205	228	engineered and purified	experimental_method	To test the aspects of the Tdp2 reaction mechanism described here derived from high-resolution mouse Tdp2 crystal structures (denoted with superscript numbering ‘m’ for numbering of the mouse protein), we engineered and purified thirteen human MBP-hTdp2cat mutant proteins (denoted with superscript numbering and ‘h’ for the human protein) and assayed the impacts of mutations on Tdp2 catalytic activity using three in vitro reporter substrates including a tyrosylated DNA substrate (5′-Y), p-nitrophenyl phosphate (PNPP) and thymidine 5′-monophosphate p-nitrophenyl ester (T5PNP) (Figure 5E, Supplementary Figures S5B and S5C).	RESULTS
238	243	human	species	To test the aspects of the Tdp2 reaction mechanism described here derived from high-resolution mouse Tdp2 crystal structures (denoted with superscript numbering ‘m’ for numbering of the mouse protein), we engineered and purified thirteen human MBP-hTdp2cat mutant proteins (denoted with superscript numbering and ‘h’ for the human protein) and assayed the impacts of mutations on Tdp2 catalytic activity using three in vitro reporter substrates including a tyrosylated DNA substrate (5′-Y), p-nitrophenyl phosphate (PNPP) and thymidine 5′-monophosphate p-nitrophenyl ester (T5PNP) (Figure 5E, Supplementary Figures S5B and S5C).	RESULTS
244	247	MBP	experimental_method	To test the aspects of the Tdp2 reaction mechanism described here derived from high-resolution mouse Tdp2 crystal structures (denoted with superscript numbering ‘m’ for numbering of the mouse protein), we engineered and purified thirteen human MBP-hTdp2cat mutant proteins (denoted with superscript numbering and ‘h’ for the human protein) and assayed the impacts of mutations on Tdp2 catalytic activity using three in vitro reporter substrates including a tyrosylated DNA substrate (5′-Y), p-nitrophenyl phosphate (PNPP) and thymidine 5′-monophosphate p-nitrophenyl ester (T5PNP) (Figure 5E, Supplementary Figures S5B and S5C).	RESULTS
248	256	hTdp2cat	structure_element	To test the aspects of the Tdp2 reaction mechanism described here derived from high-resolution mouse Tdp2 crystal structures (denoted with superscript numbering ‘m’ for numbering of the mouse protein), we engineered and purified thirteen human MBP-hTdp2cat mutant proteins (denoted with superscript numbering and ‘h’ for the human protein) and assayed the impacts of mutations on Tdp2 catalytic activity using three in vitro reporter substrates including a tyrosylated DNA substrate (5′-Y), p-nitrophenyl phosphate (PNPP) and thymidine 5′-monophosphate p-nitrophenyl ester (T5PNP) (Figure 5E, Supplementary Figures S5B and S5C).	RESULTS
257	263	mutant	protein_state	To test the aspects of the Tdp2 reaction mechanism described here derived from high-resolution mouse Tdp2 crystal structures (denoted with superscript numbering ‘m’ for numbering of the mouse protein), we engineered and purified thirteen human MBP-hTdp2cat mutant proteins (denoted with superscript numbering and ‘h’ for the human protein) and assayed the impacts of mutations on Tdp2 catalytic activity using three in vitro reporter substrates including a tyrosylated DNA substrate (5′-Y), p-nitrophenyl phosphate (PNPP) and thymidine 5′-monophosphate p-nitrophenyl ester (T5PNP) (Figure 5E, Supplementary Figures S5B and S5C).	RESULTS
325	330	human	species	To test the aspects of the Tdp2 reaction mechanism described here derived from high-resolution mouse Tdp2 crystal structures (denoted with superscript numbering ‘m’ for numbering of the mouse protein), we engineered and purified thirteen human MBP-hTdp2cat mutant proteins (denoted with superscript numbering and ‘h’ for the human protein) and assayed the impacts of mutations on Tdp2 catalytic activity using three in vitro reporter substrates including a tyrosylated DNA substrate (5′-Y), p-nitrophenyl phosphate (PNPP) and thymidine 5′-monophosphate p-nitrophenyl ester (T5PNP) (Figure 5E, Supplementary Figures S5B and S5C).	RESULTS
367	376	mutations	experimental_method	To test the aspects of the Tdp2 reaction mechanism described here derived from high-resolution mouse Tdp2 crystal structures (denoted with superscript numbering ‘m’ for numbering of the mouse protein), we engineered and purified thirteen human MBP-hTdp2cat mutant proteins (denoted with superscript numbering and ‘h’ for the human protein) and assayed the impacts of mutations on Tdp2 catalytic activity using three in vitro reporter substrates including a tyrosylated DNA substrate (5′-Y), p-nitrophenyl phosphate (PNPP) and thymidine 5′-monophosphate p-nitrophenyl ester (T5PNP) (Figure 5E, Supplementary Figures S5B and S5C).	RESULTS
380	384	Tdp2	protein	To test the aspects of the Tdp2 reaction mechanism described here derived from high-resolution mouse Tdp2 crystal structures (denoted with superscript numbering ‘m’ for numbering of the mouse protein), we engineered and purified thirteen human MBP-hTdp2cat mutant proteins (denoted with superscript numbering and ‘h’ for the human protein) and assayed the impacts of mutations on Tdp2 catalytic activity using three in vitro reporter substrates including a tyrosylated DNA substrate (5′-Y), p-nitrophenyl phosphate (PNPP) and thymidine 5′-monophosphate p-nitrophenyl ester (T5PNP) (Figure 5E, Supplementary Figures S5B and S5C).	RESULTS
457	468	tyrosylated	protein_state	To test the aspects of the Tdp2 reaction mechanism described here derived from high-resolution mouse Tdp2 crystal structures (denoted with superscript numbering ‘m’ for numbering of the mouse protein), we engineered and purified thirteen human MBP-hTdp2cat mutant proteins (denoted with superscript numbering and ‘h’ for the human protein) and assayed the impacts of mutations on Tdp2 catalytic activity using three in vitro reporter substrates including a tyrosylated DNA substrate (5′-Y), p-nitrophenyl phosphate (PNPP) and thymidine 5′-monophosphate p-nitrophenyl ester (T5PNP) (Figure 5E, Supplementary Figures S5B and S5C).	RESULTS
469	472	DNA	chemical	To test the aspects of the Tdp2 reaction mechanism described here derived from high-resolution mouse Tdp2 crystal structures (denoted with superscript numbering ‘m’ for numbering of the mouse protein), we engineered and purified thirteen human MBP-hTdp2cat mutant proteins (denoted with superscript numbering and ‘h’ for the human protein) and assayed the impacts of mutations on Tdp2 catalytic activity using three in vitro reporter substrates including a tyrosylated DNA substrate (5′-Y), p-nitrophenyl phosphate (PNPP) and thymidine 5′-monophosphate p-nitrophenyl ester (T5PNP) (Figure 5E, Supplementary Figures S5B and S5C).	RESULTS
484	488	5′-Y	ptm	To test the aspects of the Tdp2 reaction mechanism described here derived from high-resolution mouse Tdp2 crystal structures (denoted with superscript numbering ‘m’ for numbering of the mouse protein), we engineered and purified thirteen human MBP-hTdp2cat mutant proteins (denoted with superscript numbering and ‘h’ for the human protein) and assayed the impacts of mutations on Tdp2 catalytic activity using three in vitro reporter substrates including a tyrosylated DNA substrate (5′-Y), p-nitrophenyl phosphate (PNPP) and thymidine 5′-monophosphate p-nitrophenyl ester (T5PNP) (Figure 5E, Supplementary Figures S5B and S5C).	RESULTS
491	514	p-nitrophenyl phosphate	chemical	To test the aspects of the Tdp2 reaction mechanism described here derived from high-resolution mouse Tdp2 crystal structures (denoted with superscript numbering ‘m’ for numbering of the mouse protein), we engineered and purified thirteen human MBP-hTdp2cat mutant proteins (denoted with superscript numbering and ‘h’ for the human protein) and assayed the impacts of mutations on Tdp2 catalytic activity using three in vitro reporter substrates including a tyrosylated DNA substrate (5′-Y), p-nitrophenyl phosphate (PNPP) and thymidine 5′-monophosphate p-nitrophenyl ester (T5PNP) (Figure 5E, Supplementary Figures S5B and S5C).	RESULTS
516	520	PNPP	chemical	To test the aspects of the Tdp2 reaction mechanism described here derived from high-resolution mouse Tdp2 crystal structures (denoted with superscript numbering ‘m’ for numbering of the mouse protein), we engineered and purified thirteen human MBP-hTdp2cat mutant proteins (denoted with superscript numbering and ‘h’ for the human protein) and assayed the impacts of mutations on Tdp2 catalytic activity using three in vitro reporter substrates including a tyrosylated DNA substrate (5′-Y), p-nitrophenyl phosphate (PNPP) and thymidine 5′-monophosphate p-nitrophenyl ester (T5PNP) (Figure 5E, Supplementary Figures S5B and S5C).	RESULTS
526	572	thymidine 5′-monophosphate p-nitrophenyl ester	chemical	To test the aspects of the Tdp2 reaction mechanism described here derived from high-resolution mouse Tdp2 crystal structures (denoted with superscript numbering ‘m’ for numbering of the mouse protein), we engineered and purified thirteen human MBP-hTdp2cat mutant proteins (denoted with superscript numbering and ‘h’ for the human protein) and assayed the impacts of mutations on Tdp2 catalytic activity using three in vitro reporter substrates including a tyrosylated DNA substrate (5′-Y), p-nitrophenyl phosphate (PNPP) and thymidine 5′-monophosphate p-nitrophenyl ester (T5PNP) (Figure 5E, Supplementary Figures S5B and S5C).	RESULTS
574	579	T5PNP	chemical	To test the aspects of the Tdp2 reaction mechanism described here derived from high-resolution mouse Tdp2 crystal structures (denoted with superscript numbering ‘m’ for numbering of the mouse protein), we engineered and purified thirteen human MBP-hTdp2cat mutant proteins (denoted with superscript numbering and ‘h’ for the human protein) and assayed the impacts of mutations on Tdp2 catalytic activity using three in vitro reporter substrates including a tyrosylated DNA substrate (5′-Y), p-nitrophenyl phosphate (PNPP) and thymidine 5′-monophosphate p-nitrophenyl ester (T5PNP) (Figure 5E, Supplementary Figures S5B and S5C).	RESULTS
132	136	Tdp2	protein	By analyzing activities on this nested set of chemically related substrates we aimed to dissect structure-activity relationships of Tdp2 catalysis.	RESULTS
13	22	mutations	experimental_method	For example, mutations impacting Tdp2 active site chemistry and phosphotyrosyl bond cleavage should similarly affect catalysis on all three substrates, but mutants impacting DNA damage binding might only impair catalysis on 5′-Y and T5PNP but not PNPP that lacks a nucleobase.	RESULTS
33	37	Tdp2	protein	For example, mutations impacting Tdp2 active site chemistry and phosphotyrosyl bond cleavage should similarly affect catalysis on all three substrates, but mutants impacting DNA damage binding might only impair catalysis on 5′-Y and T5PNP but not PNPP that lacks a nucleobase.	RESULTS
38	49	active site	site	For example, mutations impacting Tdp2 active site chemistry and phosphotyrosyl bond cleavage should similarly affect catalysis on all three substrates, but mutants impacting DNA damage binding might only impair catalysis on 5′-Y and T5PNP but not PNPP that lacks a nucleobase.	RESULTS
64	78	phosphotyrosyl	ptm	For example, mutations impacting Tdp2 active site chemistry and phosphotyrosyl bond cleavage should similarly affect catalysis on all three substrates, but mutants impacting DNA damage binding might only impair catalysis on 5′-Y and T5PNP but not PNPP that lacks a nucleobase.	RESULTS
174	177	DNA	chemical	For example, mutations impacting Tdp2 active site chemistry and phosphotyrosyl bond cleavage should similarly affect catalysis on all three substrates, but mutants impacting DNA damage binding might only impair catalysis on 5′-Y and T5PNP but not PNPP that lacks a nucleobase.	RESULTS
224	228	5′-Y	ptm	For example, mutations impacting Tdp2 active site chemistry and phosphotyrosyl bond cleavage should similarly affect catalysis on all three substrates, but mutants impacting DNA damage binding might only impair catalysis on 5′-Y and T5PNP but not PNPP that lacks a nucleobase.	RESULTS
233	238	T5PNP	chemical	For example, mutations impacting Tdp2 active site chemistry and phosphotyrosyl bond cleavage should similarly affect catalysis on all three substrates, but mutants impacting DNA damage binding might only impair catalysis on 5′-Y and T5PNP but not PNPP that lacks a nucleobase.	RESULTS
247	251	PNPP	chemical	For example, mutations impacting Tdp2 active site chemistry and phosphotyrosyl bond cleavage should similarly affect catalysis on all three substrates, but mutants impacting DNA damage binding might only impair catalysis on 5′-Y and T5PNP but not PNPP that lacks a nucleobase.	RESULTS
0	18	Structural results	evidence	Structural results and QM/MM modeling indicate mAsp272 activates a water molecule for in-line nucleophilic attack of the scissile phosphotyrosyl linkage.	RESULTS
23	37	QM/MM modeling	experimental_method	Structural results and QM/MM modeling indicate mAsp272 activates a water molecule for in-line nucleophilic attack of the scissile phosphotyrosyl linkage.	RESULTS
47	54	mAsp272	residue_name_number	Structural results and QM/MM modeling indicate mAsp272 activates a water molecule for in-line nucleophilic attack of the scissile phosphotyrosyl linkage.	RESULTS
67	72	water	chemical	Structural results and QM/MM modeling indicate mAsp272 activates a water molecule for in-line nucleophilic attack of the scissile phosphotyrosyl linkage.	RESULTS
130	152	phosphotyrosyl linkage	ptm	Structural results and QM/MM modeling indicate mAsp272 activates a water molecule for in-line nucleophilic attack of the scissile phosphotyrosyl linkage.	RESULTS
74	81	mutated	experimental_method	To test if this proposed Lewis base is critical for reaction chemistry we mutated it to a His, which could alternatively support metal binding, as well as bulky hydrophobic residues (Leu and Met) that we predict would block the water-binding site.	RESULTS
85	87	to	experimental_method	To test if this proposed Lewis base is critical for reaction chemistry we mutated it to a His, which could alternatively support metal binding, as well as bulky hydrophobic residues (Leu and Met) that we predict would block the water-binding site.	RESULTS
90	93	His	residue_name	To test if this proposed Lewis base is critical for reaction chemistry we mutated it to a His, which could alternatively support metal binding, as well as bulky hydrophobic residues (Leu and Met) that we predict would block the water-binding site.	RESULTS
183	186	Leu	residue_name	To test if this proposed Lewis base is critical for reaction chemistry we mutated it to a His, which could alternatively support metal binding, as well as bulky hydrophobic residues (Leu and Met) that we predict would block the water-binding site.	RESULTS
191	194	Met	residue_name	To test if this proposed Lewis base is critical for reaction chemistry we mutated it to a His, which could alternatively support metal binding, as well as bulky hydrophobic residues (Leu and Met) that we predict would block the water-binding site.	RESULTS
228	246	water-binding site	site	To test if this proposed Lewis base is critical for reaction chemistry we mutated it to a His, which could alternatively support metal binding, as well as bulky hydrophobic residues (Leu and Met) that we predict would block the water-binding site.	RESULTS
38	44	hD262N	mutant	Similar to a previously characterized hD262N mutation, all three substitutions ablate activity, supporting essential roles for hAsp262 (mAsp272) in catalysis.	RESULTS
65	78	substitutions	experimental_method	Similar to a previously characterized hD262N mutation, all three substitutions ablate activity, supporting essential roles for hAsp262 (mAsp272) in catalysis.	RESULTS
127	134	hAsp262	residue_name_number	Similar to a previously characterized hD262N mutation, all three substitutions ablate activity, supporting essential roles for hAsp262 (mAsp272) in catalysis.	RESULTS
136	143	mAsp272	residue_name_number	Similar to a previously characterized hD262N mutation, all three substitutions ablate activity, supporting essential roles for hAsp262 (mAsp272) in catalysis.	RESULTS
9	16	mutated	experimental_method	Next, we mutated key elements of the mobile loop (β2Hβ hydrophobic wall, Figure 2A and C).	RESULTS
50	71	β2Hβ hydrophobic wall	site	Next, we mutated key elements of the mobile loop (β2Hβ hydrophobic wall, Figure 2A and C).	RESULTS
0	9	Mutations	experimental_method	Mutations hI307A, hL305A, hL305F and hL305W all impaired catalysis on both nucleotide-containing substrates (<50% activity).	RESULTS
10	16	hI307A	mutant	Mutations hI307A, hL305A, hL305F and hL305W all impaired catalysis on both nucleotide-containing substrates (<50% activity).	RESULTS
18	24	hL305A	mutant	Mutations hI307A, hL305A, hL305F and hL305W all impaired catalysis on both nucleotide-containing substrates (<50% activity).	RESULTS
26	32	hL305F	mutant	Mutations hI307A, hL305A, hL305F and hL305W all impaired catalysis on both nucleotide-containing substrates (<50% activity).	RESULTS
37	43	hL305W	mutant	Mutations hI307A, hL305A, hL305F and hL305W all impaired catalysis on both nucleotide-containing substrates (<50% activity).	RESULTS
4	10	hL305W	mutant	The hL305W substitution that we expect to have the most distorting impact on conformation of the β2Hβ hydrophobic wall also has the largest impact on catalysis of the DNA substrate 5′-Y. By comparison, as predicted by our model where β2Hβ dictates key interactions with undamaged and damaged nucleobases, all of these substitutions have little impact on PNPP (>90% activity).	RESULTS
97	118	β2Hβ hydrophobic wall	site	The hL305W substitution that we expect to have the most distorting impact on conformation of the β2Hβ hydrophobic wall also has the largest impact on catalysis of the DNA substrate 5′-Y. By comparison, as predicted by our model where β2Hβ dictates key interactions with undamaged and damaged nucleobases, all of these substitutions have little impact on PNPP (>90% activity).	RESULTS
167	170	DNA	chemical	The hL305W substitution that we expect to have the most distorting impact on conformation of the β2Hβ hydrophobic wall also has the largest impact on catalysis of the DNA substrate 5′-Y. By comparison, as predicted by our model where β2Hβ dictates key interactions with undamaged and damaged nucleobases, all of these substitutions have little impact on PNPP (>90% activity).	RESULTS
181	185	5′-Y	ptm	The hL305W substitution that we expect to have the most distorting impact on conformation of the β2Hβ hydrophobic wall also has the largest impact on catalysis of the DNA substrate 5′-Y. By comparison, as predicted by our model where β2Hβ dictates key interactions with undamaged and damaged nucleobases, all of these substitutions have little impact on PNPP (>90% activity).	RESULTS
234	238	β2Hβ	structure_element	The hL305W substitution that we expect to have the most distorting impact on conformation of the β2Hβ hydrophobic wall also has the largest impact on catalysis of the DNA substrate 5′-Y. By comparison, as predicted by our model where β2Hβ dictates key interactions with undamaged and damaged nucleobases, all of these substitutions have little impact on PNPP (>90% activity).	RESULTS
318	331	substitutions	experimental_method	The hL305W substitution that we expect to have the most distorting impact on conformation of the β2Hβ hydrophobic wall also has the largest impact on catalysis of the DNA substrate 5′-Y. By comparison, as predicted by our model where β2Hβ dictates key interactions with undamaged and damaged nucleobases, all of these substitutions have little impact on PNPP (>90% activity).	RESULTS
354	358	PNPP	chemical	The hL305W substitution that we expect to have the most distorting impact on conformation of the β2Hβ hydrophobic wall also has the largest impact on catalysis of the DNA substrate 5′-Y. By comparison, as predicted by our model where β2Hβ dictates key interactions with undamaged and damaged nucleobases, all of these substitutions have little impact on PNPP (>90% activity).	RESULTS
45	80	enzyme substrate cation–π interface	site	Third, we altered properties of the proposed enzyme substrate cation–π interface.	RESULTS
31	37	mutant	protein_state	No activity was detected for a mutant that removes the positive charge at this position (hR206A).	RESULTS
89	95	hR206A	mutant	No activity was detected for a mutant that removes the positive charge at this position (hR206A).	RESULTS
29	35	pocket	site	The precise geometry of this pocket is also critical for catalysis as replacement of hArg206 (mArg216) with a lysine also results in a profound decrease in catalysis (<5% activity on 5′-Y, no detectable activity on T5PNP or PNPP).	RESULTS
70	81	replacement	experimental_method	The precise geometry of this pocket is also critical for catalysis as replacement of hArg206 (mArg216) with a lysine also results in a profound decrease in catalysis (<5% activity on 5′-Y, no detectable activity on T5PNP or PNPP).	RESULTS
85	92	hArg206	residue_name_number	The precise geometry of this pocket is also critical for catalysis as replacement of hArg206 (mArg216) with a lysine also results in a profound decrease in catalysis (<5% activity on 5′-Y, no detectable activity on T5PNP or PNPP).	RESULTS
94	101	mArg216	residue_name_number	The precise geometry of this pocket is also critical for catalysis as replacement of hArg206 (mArg216) with a lysine also results in a profound decrease in catalysis (<5% activity on 5′-Y, no detectable activity on T5PNP or PNPP).	RESULTS
110	116	lysine	residue_name	The precise geometry of this pocket is also critical for catalysis as replacement of hArg206 (mArg216) with a lysine also results in a profound decrease in catalysis (<5% activity on 5′-Y, no detectable activity on T5PNP or PNPP).	RESULTS
183	187	5′-Y	ptm	The precise geometry of this pocket is also critical for catalysis as replacement of hArg206 (mArg216) with a lysine also results in a profound decrease in catalysis (<5% activity on 5′-Y, no detectable activity on T5PNP or PNPP).	RESULTS
215	220	T5PNP	chemical	The precise geometry of this pocket is also critical for catalysis as replacement of hArg206 (mArg216) with a lysine also results in a profound decrease in catalysis (<5% activity on 5′-Y, no detectable activity on T5PNP or PNPP).	RESULTS
224	228	PNPP	chemical	The precise geometry of this pocket is also critical for catalysis as replacement of hArg206 (mArg216) with a lysine also results in a profound decrease in catalysis (<5% activity on 5′-Y, no detectable activity on T5PNP or PNPP).	RESULTS
11	19	mutation	experimental_method	Similarly, mutation of hTyr178 that structurally scaffolds the hArg206 (mArg216) guanidinium also significantly impacts activity, with Y178F and Y178W having <25% activity on all substrates.	RESULTS
23	30	hTyr178	residue_name_number	Similarly, mutation of hTyr178 that structurally scaffolds the hArg206 (mArg216) guanidinium also significantly impacts activity, with Y178F and Y178W having <25% activity on all substrates.	RESULTS
63	70	hArg206	residue_name_number	Similarly, mutation of hTyr178 that structurally scaffolds the hArg206 (mArg216) guanidinium also significantly impacts activity, with Y178F and Y178W having <25% activity on all substrates.	RESULTS
72	79	mArg216	residue_name_number	Similarly, mutation of hTyr178 that structurally scaffolds the hArg206 (mArg216) guanidinium also significantly impacts activity, with Y178F and Y178W having <25% activity on all substrates.	RESULTS
135	140	Y178F	mutant	Similarly, mutation of hTyr178 that structurally scaffolds the hArg206 (mArg216) guanidinium also significantly impacts activity, with Y178F and Y178W having <25% activity on all substrates.	RESULTS
145	150	Y178W	mutant	Similarly, mutation of hTyr178 that structurally scaffolds the hArg206 (mArg216) guanidinium also significantly impacts activity, with Y178F and Y178W having <25% activity on all substrates.	RESULTS
35	42	hHis351	residue_name_number	Fourth, we evaluated roles for the hHis351–hAsp316 (mAsp326–mHis359) transition state stabilization charge pair.	RESULTS
43	50	hAsp316	residue_name_number	Fourth, we evaluated roles for the hHis351–hAsp316 (mAsp326–mHis359) transition state stabilization charge pair.	RESULTS
52	59	mAsp326	residue_name_number	Fourth, we evaluated roles for the hHis351–hAsp316 (mAsp326–mHis359) transition state stabilization charge pair.	RESULTS
60	67	mHis359	residue_name_number	Fourth, we evaluated roles for the hHis351–hAsp316 (mAsp326–mHis359) transition state stabilization charge pair.	RESULTS
14	23	mutations	experimental_method	We found that mutations that removed the charge yet retained the ability to hydrogen bond (hH351Q) or should abrogate the elevated pKa of the Histidine (hD316N) had severe impacts on catalysis.	RESULTS
29	36	removed	experimental_method	We found that mutations that removed the charge yet retained the ability to hydrogen bond (hH351Q) or should abrogate the elevated pKa of the Histidine (hD316N) had severe impacts on catalysis.	RESULTS
76	89	hydrogen bond	bond_interaction	We found that mutations that removed the charge yet retained the ability to hydrogen bond (hH351Q) or should abrogate the elevated pKa of the Histidine (hD316N) had severe impacts on catalysis.	RESULTS
91	97	hH351Q	mutant	We found that mutations that removed the charge yet retained the ability to hydrogen bond (hH351Q) or should abrogate the elevated pKa of the Histidine (hD316N) had severe impacts on catalysis.	RESULTS
131	134	pKa	evidence	We found that mutations that removed the charge yet retained the ability to hydrogen bond (hH351Q) or should abrogate the elevated pKa of the Histidine (hD316N) had severe impacts on catalysis.	RESULTS
142	151	Histidine	residue_name	We found that mutations that removed the charge yet retained the ability to hydrogen bond (hH351Q) or should abrogate the elevated pKa of the Histidine (hD316N) had severe impacts on catalysis.	RESULTS
153	159	hD316N	mutant	We found that mutations that removed the charge yet retained the ability to hydrogen bond (hH351Q) or should abrogate the elevated pKa of the Histidine (hD316N) had severe impacts on catalysis.	RESULTS
63	74	active site	site	Thus altogether, our mutational data support key roles for the active site Lewis base aspartate, mobile substrate engagement loops, enzyme–substrate cation–π interactions, and active site transition state stabilizing charge interaction in supporting Tdp2 catalysis.	RESULTS
86	95	aspartate	residue_name	Thus altogether, our mutational data support key roles for the active site Lewis base aspartate, mobile substrate engagement loops, enzyme–substrate cation–π interactions, and active site transition state stabilizing charge interaction in supporting Tdp2 catalysis.	RESULTS
97	103	mobile	protein_state	Thus altogether, our mutational data support key roles for the active site Lewis base aspartate, mobile substrate engagement loops, enzyme–substrate cation–π interactions, and active site transition state stabilizing charge interaction in supporting Tdp2 catalysis.	RESULTS
104	130	substrate engagement loops	structure_element	Thus altogether, our mutational data support key roles for the active site Lewis base aspartate, mobile substrate engagement loops, enzyme–substrate cation–π interactions, and active site transition state stabilizing charge interaction in supporting Tdp2 catalysis.	RESULTS
149	170	cation–π interactions	bond_interaction	Thus altogether, our mutational data support key roles for the active site Lewis base aspartate, mobile substrate engagement loops, enzyme–substrate cation–π interactions, and active site transition state stabilizing charge interaction in supporting Tdp2 catalysis.	RESULTS
176	187	active site	site	Thus altogether, our mutational data support key roles for the active site Lewis base aspartate, mobile substrate engagement loops, enzyme–substrate cation–π interactions, and active site transition state stabilizing charge interaction in supporting Tdp2 catalysis.	RESULTS
217	235	charge interaction	bond_interaction	Thus altogether, our mutational data support key roles for the active site Lewis base aspartate, mobile substrate engagement loops, enzyme–substrate cation–π interactions, and active site transition state stabilizing charge interaction in supporting Tdp2 catalysis.	RESULTS
250	254	Tdp2	protein	Thus altogether, our mutational data support key roles for the active site Lewis base aspartate, mobile substrate engagement loops, enzyme–substrate cation–π interactions, and active site transition state stabilizing charge interaction in supporting Tdp2 catalysis.	RESULTS
2	6	Tdp2	protein	A Tdp2 active site single nucleotide polymorphism impairs Tdp2 function	RESULTS
7	18	active site	site	A Tdp2 active site single nucleotide polymorphism impairs Tdp2 function	RESULTS
58	62	Tdp2	protein	A Tdp2 active site single nucleotide polymorphism impairs Tdp2 function	RESULTS
44	48	TDP2	protein	Recently, it was found that inactivation of TDP2 by a splice-site mutation is associated with neurological disease and confers hypersensitivity to Top2 poisons.	RESULTS
147	151	Top2	protein_type	Recently, it was found that inactivation of TDP2 by a splice-site mutation is associated with neurological disease and confers hypersensitivity to Top2 poisons.	RESULTS
22	27	human	species	We considered whether human SNPs causing missense mutations might also impact Tdp2 DNA–protein crosslink repair functions established here as well as Tdp2-mediated NHEJ of blocked DNA termini.	RESULTS
78	82	Tdp2	protein	We considered whether human SNPs causing missense mutations might also impact Tdp2 DNA–protein crosslink repair functions established here as well as Tdp2-mediated NHEJ of blocked DNA termini.	RESULTS
83	86	DNA	chemical	We considered whether human SNPs causing missense mutations might also impact Tdp2 DNA–protein crosslink repair functions established here as well as Tdp2-mediated NHEJ of blocked DNA termini.	RESULTS
150	154	Tdp2	protein	We considered whether human SNPs causing missense mutations might also impact Tdp2 DNA–protein crosslink repair functions established here as well as Tdp2-mediated NHEJ of blocked DNA termini.	RESULTS
180	183	DNA	chemical	We considered whether human SNPs causing missense mutations might also impact Tdp2 DNA–protein crosslink repair functions established here as well as Tdp2-mediated NHEJ of blocked DNA termini.	RESULTS
26	31	human	species	We identified two SNPs in human TDP2 curated in the NCBI SNP database that result in missense mutations within the DNA processing active site: rs199602263 (minor allele frequency 0.0002), which substitutes hAsp350 for Asn, and rs77273535 (minor allele frequency 0.004, which substitutes hIle307 for Val) (Figure 6A).	RESULTS
32	36	TDP2	protein	We identified two SNPs in human TDP2 curated in the NCBI SNP database that result in missense mutations within the DNA processing active site: rs199602263 (minor allele frequency 0.0002), which substitutes hAsp350 for Asn, and rs77273535 (minor allele frequency 0.004, which substitutes hIle307 for Val) (Figure 6A).	RESULTS
115	141	DNA processing active site	site	We identified two SNPs in human TDP2 curated in the NCBI SNP database that result in missense mutations within the DNA processing active site: rs199602263 (minor allele frequency 0.0002), which substitutes hAsp350 for Asn, and rs77273535 (minor allele frequency 0.004, which substitutes hIle307 for Val) (Figure 6A).	RESULTS
143	154	rs199602263	gene	We identified two SNPs in human TDP2 curated in the NCBI SNP database that result in missense mutations within the DNA processing active site: rs199602263 (minor allele frequency 0.0002), which substitutes hAsp350 for Asn, and rs77273535 (minor allele frequency 0.004, which substitutes hIle307 for Val) (Figure 6A).	RESULTS
206	213	hAsp350	residue_name_number	We identified two SNPs in human TDP2 curated in the NCBI SNP database that result in missense mutations within the DNA processing active site: rs199602263 (minor allele frequency 0.0002), which substitutes hAsp350 for Asn, and rs77273535 (minor allele frequency 0.004, which substitutes hIle307 for Val) (Figure 6A).	RESULTS
218	221	Asn	residue_name	We identified two SNPs in human TDP2 curated in the NCBI SNP database that result in missense mutations within the DNA processing active site: rs199602263 (minor allele frequency 0.0002), which substitutes hAsp350 for Asn, and rs77273535 (minor allele frequency 0.004, which substitutes hIle307 for Val) (Figure 6A).	RESULTS
227	237	rs77273535	gene	We identified two SNPs in human TDP2 curated in the NCBI SNP database that result in missense mutations within the DNA processing active site: rs199602263 (minor allele frequency 0.0002), which substitutes hAsp350 for Asn, and rs77273535 (minor allele frequency 0.004, which substitutes hIle307 for Val) (Figure 6A).	RESULTS
287	294	hIle307	residue_name_number	We identified two SNPs in human TDP2 curated in the NCBI SNP database that result in missense mutations within the DNA processing active site: rs199602263 (minor allele frequency 0.0002), which substitutes hAsp350 for Asn, and rs77273535 (minor allele frequency 0.004, which substitutes hIle307 for Val) (Figure 6A).	RESULTS
299	302	Val	residue_name	We identified two SNPs in human TDP2 curated in the NCBI SNP database that result in missense mutations within the DNA processing active site: rs199602263 (minor allele frequency 0.0002), which substitutes hAsp350 for Asn, and rs77273535 (minor allele frequency 0.004, which substitutes hIle307 for Val) (Figure 6A).	RESULTS
12	18	hD350N	mutant	We show the hD350N substitution severely impairs activity on all substrates tested in vitro, whereas hI307V only has a mild impact on catalysis (Figure 6B–D).	RESULTS
19	31	substitution	experimental_method	We show the hD350N substitution severely impairs activity on all substrates tested in vitro, whereas hI307V only has a mild impact on catalysis (Figure 6B–D).	RESULTS
101	107	hI307V	mutant	We show the hD350N substitution severely impairs activity on all substrates tested in vitro, whereas hI307V only has a mild impact on catalysis (Figure 6B–D).	RESULTS
39	44	D350N	mutant	To better understand the basis for the D350N catalytic defect, we analyzed the structural environment of this substitution based on the high-resolution structures of mTdp2cat (Figure 6A).	RESULTS
152	162	structures	evidence	To better understand the basis for the D350N catalytic defect, we analyzed the structural environment of this substitution based on the high-resolution structures of mTdp2cat (Figure 6A).	RESULTS
166	174	mTdp2cat	structure_element	To better understand the basis for the D350N catalytic defect, we analyzed the structural environment of this substitution based on the high-resolution structures of mTdp2cat (Figure 6A).	RESULTS
19	23	Tdp2	protein	Interestingly, the Tdp2 single Mg2+ ion octahedral coordination shell also involves an extended hydrogen-bonding network mediated by hAsp350 (mAsp358) that stabilizes the DNA-bound conformation of the β2Hβ substrate-binding loop through hydrogen bonding to mTrp307.	RESULTS
31	35	Mg2+	chemical	Interestingly, the Tdp2 single Mg2+ ion octahedral coordination shell also involves an extended hydrogen-bonding network mediated by hAsp350 (mAsp358) that stabilizes the DNA-bound conformation of the β2Hβ substrate-binding loop through hydrogen bonding to mTrp307.	RESULTS
40	69	octahedral coordination shell	bond_interaction	Interestingly, the Tdp2 single Mg2+ ion octahedral coordination shell also involves an extended hydrogen-bonding network mediated by hAsp350 (mAsp358) that stabilizes the DNA-bound conformation of the β2Hβ substrate-binding loop through hydrogen bonding to mTrp307.	RESULTS
96	120	hydrogen-bonding network	bond_interaction	Interestingly, the Tdp2 single Mg2+ ion octahedral coordination shell also involves an extended hydrogen-bonding network mediated by hAsp350 (mAsp358) that stabilizes the DNA-bound conformation of the β2Hβ substrate-binding loop through hydrogen bonding to mTrp307.	RESULTS
133	140	hAsp350	residue_name_number	Interestingly, the Tdp2 single Mg2+ ion octahedral coordination shell also involves an extended hydrogen-bonding network mediated by hAsp350 (mAsp358) that stabilizes the DNA-bound conformation of the β2Hβ substrate-binding loop through hydrogen bonding to mTrp307.	RESULTS
142	149	mAsp358	residue_name_number	Interestingly, the Tdp2 single Mg2+ ion octahedral coordination shell also involves an extended hydrogen-bonding network mediated by hAsp350 (mAsp358) that stabilizes the DNA-bound conformation of the β2Hβ substrate-binding loop through hydrogen bonding to mTrp307.	RESULTS
171	180	DNA-bound	protein_state	Interestingly, the Tdp2 single Mg2+ ion octahedral coordination shell also involves an extended hydrogen-bonding network mediated by hAsp350 (mAsp358) that stabilizes the DNA-bound conformation of the β2Hβ substrate-binding loop through hydrogen bonding to mTrp307.	RESULTS
201	228	β2Hβ substrate-binding loop	structure_element	Interestingly, the Tdp2 single Mg2+ ion octahedral coordination shell also involves an extended hydrogen-bonding network mediated by hAsp350 (mAsp358) that stabilizes the DNA-bound conformation of the β2Hβ substrate-binding loop through hydrogen bonding to mTrp307.	RESULTS
237	253	hydrogen bonding	bond_interaction	Interestingly, the Tdp2 single Mg2+ ion octahedral coordination shell also involves an extended hydrogen-bonding network mediated by hAsp350 (mAsp358) that stabilizes the DNA-bound conformation of the β2Hβ substrate-binding loop through hydrogen bonding to mTrp307.	RESULTS
257	264	mTrp307	residue_name_number	Interestingly, the Tdp2 single Mg2+ ion octahedral coordination shell also involves an extended hydrogen-bonding network mediated by hAsp350 (mAsp358) that stabilizes the DNA-bound conformation of the β2Hβ substrate-binding loop through hydrogen bonding to mTrp307.	RESULTS
6	13	hAsp350	residue_name_number	Here, hAsp350 (mAsp358) serves as a structural nexus linking active site metal binding to substrate binding loop conformations.	RESULTS
15	22	mAsp358	residue_name_number	Here, hAsp350 (mAsp358) serves as a structural nexus linking active site metal binding to substrate binding loop conformations.	RESULTS
61	72	active site	site	Here, hAsp350 (mAsp358) serves as a structural nexus linking active site metal binding to substrate binding loop conformations.	RESULTS
90	112	substrate binding loop	structure_element	Here, hAsp350 (mAsp358) serves as a structural nexus linking active site metal binding to substrate binding loop conformations.	RESULTS
0	4	Tdp2	protein	Tdp2 SNPs impair function. (A) Active site residues mutated by TDP2 SNPs.	FIG
31	42	Active site	site	Tdp2 SNPs impair function. (A) Active site residues mutated by TDP2 SNPs.	FIG
63	67	TDP2	protein	Tdp2 SNPs impair function. (A) Active site residues mutated by TDP2 SNPs.	FIG
0	5	D350N	mutant	D350N (mTdp2 D358N) and I307V (mTdp2 I317V) substitutions are mapped onto the Tdp2 active site of the high-resolution mTdp2cat structure (4GZ1).	FIG
7	12	mTdp2	protein	D350N (mTdp2 D358N) and I307V (mTdp2 I317V) substitutions are mapped onto the Tdp2 active site of the high-resolution mTdp2cat structure (4GZ1).	FIG
13	18	D358N	mutant	D350N (mTdp2 D358N) and I307V (mTdp2 I317V) substitutions are mapped onto the Tdp2 active site of the high-resolution mTdp2cat structure (4GZ1).	FIG
24	29	I307V	mutant	D350N (mTdp2 D358N) and I307V (mTdp2 I317V) substitutions are mapped onto the Tdp2 active site of the high-resolution mTdp2cat structure (4GZ1).	FIG
31	36	mTdp2	protein	D350N (mTdp2 D358N) and I307V (mTdp2 I317V) substitutions are mapped onto the Tdp2 active site of the high-resolution mTdp2cat structure (4GZ1).	FIG
37	42	I317V	mutant	D350N (mTdp2 D358N) and I307V (mTdp2 I317V) substitutions are mapped onto the Tdp2 active site of the high-resolution mTdp2cat structure (4GZ1).	FIG
44	57	substitutions	experimental_method	D350N (mTdp2 D358N) and I307V (mTdp2 I317V) substitutions are mapped onto the Tdp2 active site of the high-resolution mTdp2cat structure (4GZ1).	FIG
78	82	Tdp2	protein	D350N (mTdp2 D358N) and I307V (mTdp2 I317V) substitutions are mapped onto the Tdp2 active site of the high-resolution mTdp2cat structure (4GZ1).	FIG
83	94	active site	site	D350N (mTdp2 D358N) and I307V (mTdp2 I317V) substitutions are mapped onto the Tdp2 active site of the high-resolution mTdp2cat structure (4GZ1).	FIG
118	126	mTdp2cat	structure_element	D350N (mTdp2 D358N) and I307V (mTdp2 I317V) substitutions are mapped onto the Tdp2 active site of the high-resolution mTdp2cat structure (4GZ1).	FIG
127	136	structure	evidence	D350N (mTdp2 D358N) and I307V (mTdp2 I317V) substitutions are mapped onto the Tdp2 active site of the high-resolution mTdp2cat structure (4GZ1).	FIG
27	35	SDS-PAGE	experimental_method	(B) Coomassie blue stained SDS-PAGE gel of purified WT and mutant MBP-hTdp2cat proteins used for assays in panels C and D. (C) Activity of WT and mutant MBP-hTdp2cat proteins on a 5′–phosphotyrosyl–DNA oligonucleotides with 3′-fluorescein label.	FIG
52	54	WT	protein_state	(B) Coomassie blue stained SDS-PAGE gel of purified WT and mutant MBP-hTdp2cat proteins used for assays in panels C and D. (C) Activity of WT and mutant MBP-hTdp2cat proteins on a 5′–phosphotyrosyl–DNA oligonucleotides with 3′-fluorescein label.	FIG
59	65	mutant	protein_state	(B) Coomassie blue stained SDS-PAGE gel of purified WT and mutant MBP-hTdp2cat proteins used for assays in panels C and D. (C) Activity of WT and mutant MBP-hTdp2cat proteins on a 5′–phosphotyrosyl–DNA oligonucleotides with 3′-fluorescein label.	FIG
66	69	MBP	experimental_method	(B) Coomassie blue stained SDS-PAGE gel of purified WT and mutant MBP-hTdp2cat proteins used for assays in panels C and D. (C) Activity of WT and mutant MBP-hTdp2cat proteins on a 5′–phosphotyrosyl–DNA oligonucleotides with 3′-fluorescein label.	FIG
70	78	hTdp2cat	structure_element	(B) Coomassie blue stained SDS-PAGE gel of purified WT and mutant MBP-hTdp2cat proteins used for assays in panels C and D. (C) Activity of WT and mutant MBP-hTdp2cat proteins on a 5′–phosphotyrosyl–DNA oligonucleotides with 3′-fluorescein label.	FIG
139	141	WT	protein_state	(B) Coomassie blue stained SDS-PAGE gel of purified WT and mutant MBP-hTdp2cat proteins used for assays in panels C and D. (C) Activity of WT and mutant MBP-hTdp2cat proteins on a 5′–phosphotyrosyl–DNA oligonucleotides with 3′-fluorescein label.	FIG
146	152	mutant	protein_state	(B) Coomassie blue stained SDS-PAGE gel of purified WT and mutant MBP-hTdp2cat proteins used for assays in panels C and D. (C) Activity of WT and mutant MBP-hTdp2cat proteins on a 5′–phosphotyrosyl–DNA oligonucleotides with 3′-fluorescein label.	FIG
153	156	MBP	experimental_method	(B) Coomassie blue stained SDS-PAGE gel of purified WT and mutant MBP-hTdp2cat proteins used for assays in panels C and D. (C) Activity of WT and mutant MBP-hTdp2cat proteins on a 5′–phosphotyrosyl–DNA oligonucleotides with 3′-fluorescein label.	FIG
157	165	hTdp2cat	structure_element	(B) Coomassie blue stained SDS-PAGE gel of purified WT and mutant MBP-hTdp2cat proteins used for assays in panels C and D. (C) Activity of WT and mutant MBP-hTdp2cat proteins on a 5′–phosphotyrosyl–DNA oligonucleotides with 3′-fluorescein label.	FIG
180	218	5′–phosphotyrosyl–DNA oligonucleotides	chemical	(B) Coomassie blue stained SDS-PAGE gel of purified WT and mutant MBP-hTdp2cat proteins used for assays in panels C and D. (C) Activity of WT and mutant MBP-hTdp2cat proteins on a 5′–phosphotyrosyl–DNA oligonucleotides with 3′-fluorescein label.	FIG
227	238	fluorescein	chemical	(B) Coomassie blue stained SDS-PAGE gel of purified WT and mutant MBP-hTdp2cat proteins used for assays in panels C and D. (C) Activity of WT and mutant MBP-hTdp2cat proteins on a 5′–phosphotyrosyl–DNA oligonucleotides with 3′-fluorescein label.	FIG
135	148	TBE-urea PAGE	experimental_method	Samples were withdrawn from reactions, neutralized with TBE-urea loading dye at the indicated timepoints, and electrophoresed on a 20% TBE-urea PAGE.	FIG
25	27	WT	protein_state	(D) Relative activity of WT and indicated mutant human MBP-hTdp2cat fusion proteins on three model Tdp2 substrates.	FIG
42	48	mutant	protein_state	(D) Relative activity of WT and indicated mutant human MBP-hTdp2cat fusion proteins on three model Tdp2 substrates.	FIG
49	54	human	species	(D) Relative activity of WT and indicated mutant human MBP-hTdp2cat fusion proteins on three model Tdp2 substrates.	FIG
55	58	MBP	experimental_method	(D) Relative activity of WT and indicated mutant human MBP-hTdp2cat fusion proteins on three model Tdp2 substrates.	FIG
59	67	hTdp2cat	structure_element	(D) Relative activity of WT and indicated mutant human MBP-hTdp2cat fusion proteins on three model Tdp2 substrates.	FIG
99	103	Tdp2	protein	(D) Relative activity of WT and indicated mutant human MBP-hTdp2cat fusion proteins on three model Tdp2 substrates.	FIG
26	29	MBP	experimental_method	Quantification of percent MBP-hTdp2cat activity relative to WT protein for the 5′-Y DNA oligonucleotide substrate (blue bars), T5PNP (red bars) and PNPP (green bars) is displayed.	FIG
30	38	hTdp2cat	structure_element	Quantification of percent MBP-hTdp2cat activity relative to WT protein for the 5′-Y DNA oligonucleotide substrate (blue bars), T5PNP (red bars) and PNPP (green bars) is displayed.	FIG
60	62	WT	protein_state	Quantification of percent MBP-hTdp2cat activity relative to WT protein for the 5′-Y DNA oligonucleotide substrate (blue bars), T5PNP (red bars) and PNPP (green bars) is displayed.	FIG
79	103	5′-Y DNA oligonucleotide	chemical	Quantification of percent MBP-hTdp2cat activity relative to WT protein for the 5′-Y DNA oligonucleotide substrate (blue bars), T5PNP (red bars) and PNPP (green bars) is displayed.	FIG
127	132	T5PNP	chemical	Quantification of percent MBP-hTdp2cat activity relative to WT protein for the 5′-Y DNA oligonucleotide substrate (blue bars), T5PNP (red bars) and PNPP (green bars) is displayed.	FIG
148	152	PNPP	chemical	Quantification of percent MBP-hTdp2cat activity relative to WT protein for the 5′-Y DNA oligonucleotide substrate (blue bars), T5PNP (red bars) and PNPP (green bars) is displayed.	FIG
11	14	PNP	chemical	Release of PNP from PNP phosphate (PNPP) and was detected as an increase in absorbance at 415 nm, whereas the 5′-Y substrate is quantification of activity in a gel based assay shown in Figure 6C.	FIG
20	33	PNP phosphate	chemical	Release of PNP from PNP phosphate (PNPP) and was detected as an increase in absorbance at 415 nm, whereas the 5′-Y substrate is quantification of activity in a gel based assay shown in Figure 6C.	FIG
35	39	PNPP	chemical	Release of PNP from PNP phosphate (PNPP) and was detected as an increase in absorbance at 415 nm, whereas the 5′-Y substrate is quantification of activity in a gel based assay shown in Figure 6C.	FIG
110	114	5′-Y	ptm	Release of PNP from PNP phosphate (PNPP) and was detected as an increase in absorbance at 415 nm, whereas the 5′-Y substrate is quantification of activity in a gel based assay shown in Figure 6C.	FIG
160	175	gel based assay	experimental_method	Release of PNP from PNP phosphate (PNPP) and was detected as an increase in absorbance at 415 nm, whereas the 5′-Y substrate is quantification of activity in a gel based assay shown in Figure 6C.	FIG
38	44	hD350N	mutant	To define the molecular basis for the hD350N (mD358N) defect, we crystallized and determined the structure of the DNA-free form of the mD358N protein to 2.8Å resolution (PDB entry 5INN).	RESULTS
46	52	mD358N	mutant	To define the molecular basis for the hD350N (mD358N) defect, we crystallized and determined the structure of the DNA-free form of the mD358N protein to 2.8Å resolution (PDB entry 5INN).	RESULTS
65	92	crystallized and determined	experimental_method	To define the molecular basis for the hD350N (mD358N) defect, we crystallized and determined the structure of the DNA-free form of the mD358N protein to 2.8Å resolution (PDB entry 5INN).	RESULTS
97	106	structure	evidence	To define the molecular basis for the hD350N (mD358N) defect, we crystallized and determined the structure of the DNA-free form of the mD358N protein to 2.8Å resolution (PDB entry 5INN).	RESULTS
114	122	DNA-free	protein_state	To define the molecular basis for the hD350N (mD358N) defect, we crystallized and determined the structure of the DNA-free form of the mD358N protein to 2.8Å resolution (PDB entry 5INN).	RESULTS
135	141	mD358N	mutant	To define the molecular basis for the hD350N (mD358N) defect, we crystallized and determined the structure of the DNA-free form of the mD358N protein to 2.8Å resolution (PDB entry 5INN).	RESULTS
5	14	structure	evidence	This structure shows the D358N mutation disrupts the hydrogen bond between Asp358 and Trp307, shifts the position of Asn358 and destabilizes Trp307.	RESULTS
25	30	D358N	mutant	This structure shows the D358N mutation disrupts the hydrogen bond between Asp358 and Trp307, shifts the position of Asn358 and destabilizes Trp307.	RESULTS
31	39	mutation	experimental_method	This structure shows the D358N mutation disrupts the hydrogen bond between Asp358 and Trp307, shifts the position of Asn358 and destabilizes Trp307.	RESULTS
53	66	hydrogen bond	bond_interaction	This structure shows the D358N mutation disrupts the hydrogen bond between Asp358 and Trp307, shifts the position of Asn358 and destabilizes Trp307.	RESULTS
75	81	Asp358	residue_name_number	This structure shows the D358N mutation disrupts the hydrogen bond between Asp358 and Trp307, shifts the position of Asn358 and destabilizes Trp307.	RESULTS
86	92	Trp307	residue_name_number	This structure shows the D358N mutation disrupts the hydrogen bond between Asp358 and Trp307, shifts the position of Asn358 and destabilizes Trp307.	RESULTS
117	123	Asn358	residue_name_number	This structure shows the D358N mutation disrupts the hydrogen bond between Asp358 and Trp307, shifts the position of Asn358 and destabilizes Trp307.	RESULTS
141	147	Trp307	residue_name_number	This structure shows the D358N mutation disrupts the hydrogen bond between Asp358 and Trp307, shifts the position of Asn358 and destabilizes Trp307.	RESULTS
19	35	electron density	evidence	Consequently, poor electron density is visible for the β2Hβ loop which is mostly disordered (Supplementary Figure S6).	RESULTS
55	64	β2Hβ loop	structure_element	Consequently, poor electron density is visible for the β2Hβ loop which is mostly disordered (Supplementary Figure S6).	RESULTS
81	91	disordered	protein_state	Consequently, poor electron density is visible for the β2Hβ loop which is mostly disordered (Supplementary Figure S6).	RESULTS
9	13	Mg2+	chemical	Although Mg2+ is present at the same concentration as the WT-mTdpcat crystals (10 mM), we find the metal site is unoccupied in the mD358N crystals.	RESULTS
58	60	WT	protein_state	Although Mg2+ is present at the same concentration as the WT-mTdpcat crystals (10 mM), we find the metal site is unoccupied in the mD358N crystals.	RESULTS
61	68	mTdpcat	protein	Although Mg2+ is present at the same concentration as the WT-mTdpcat crystals (10 mM), we find the metal site is unoccupied in the mD358N crystals.	RESULTS
69	77	crystals	evidence	Although Mg2+ is present at the same concentration as the WT-mTdpcat crystals (10 mM), we find the metal site is unoccupied in the mD358N crystals.	RESULTS
99	109	metal site	site	Although Mg2+ is present at the same concentration as the WT-mTdpcat crystals (10 mM), we find the metal site is unoccupied in the mD358N crystals.	RESULTS
113	123	unoccupied	protein_state	Although Mg2+ is present at the same concentration as the WT-mTdpcat crystals (10 mM), we find the metal site is unoccupied in the mD358N crystals.	RESULTS
131	137	mD358N	mutant	Although Mg2+ is present at the same concentration as the WT-mTdpcat crystals (10 mM), we find the metal site is unoccupied in the mD358N crystals.	RESULTS
138	146	crystals	evidence	Although Mg2+ is present at the same concentration as the WT-mTdpcat crystals (10 mM), we find the metal site is unoccupied in the mD358N crystals.	RESULTS
50	61	active site	site	Therefore, metal-regulated opening/closure of the active site may modulate Tdp2 activity, and D350N is sufficient to block both metal binding and conformational change.	RESULTS
75	79	Tdp2	protein	Therefore, metal-regulated opening/closure of the active site may modulate Tdp2 activity, and D350N is sufficient to block both metal binding and conformational change.	RESULTS
94	99	D350N	mutant	Therefore, metal-regulated opening/closure of the active site may modulate Tdp2 activity, and D350N is sufficient to block both metal binding and conformational change.	RESULTS
38	44	hD350N	mutant	In support of this, we also find that hD350N (mD358N) impairs Mg2+ binding as measured by intrinsic tryptophan fluorescence (Figure 4A), and abrogates Mg2+-stimulated active site conformational changes detected by trypsin and chymotrypsin sensitivity of the Tdp2 metamorphic loop (Figure 3D).	RESULTS
46	52	mD358N	mutant	In support of this, we also find that hD350N (mD358N) impairs Mg2+ binding as measured by intrinsic tryptophan fluorescence (Figure 4A), and abrogates Mg2+-stimulated active site conformational changes detected by trypsin and chymotrypsin sensitivity of the Tdp2 metamorphic loop (Figure 3D).	RESULTS
62	66	Mg2+	chemical	In support of this, we also find that hD350N (mD358N) impairs Mg2+ binding as measured by intrinsic tryptophan fluorescence (Figure 4A), and abrogates Mg2+-stimulated active site conformational changes detected by trypsin and chymotrypsin sensitivity of the Tdp2 metamorphic loop (Figure 3D).	RESULTS
90	123	intrinsic tryptophan fluorescence	evidence	In support of this, we also find that hD350N (mD358N) impairs Mg2+ binding as measured by intrinsic tryptophan fluorescence (Figure 4A), and abrogates Mg2+-stimulated active site conformational changes detected by trypsin and chymotrypsin sensitivity of the Tdp2 metamorphic loop (Figure 3D).	RESULTS
151	155	Mg2+	chemical	In support of this, we also find that hD350N (mD358N) impairs Mg2+ binding as measured by intrinsic tryptophan fluorescence (Figure 4A), and abrogates Mg2+-stimulated active site conformational changes detected by trypsin and chymotrypsin sensitivity of the Tdp2 metamorphic loop (Figure 3D).	RESULTS
167	178	active site	site	In support of this, we also find that hD350N (mD358N) impairs Mg2+ binding as measured by intrinsic tryptophan fluorescence (Figure 4A), and abrogates Mg2+-stimulated active site conformational changes detected by trypsin and chymotrypsin sensitivity of the Tdp2 metamorphic loop (Figure 3D).	RESULTS
258	262	Tdp2	protein	In support of this, we also find that hD350N (mD358N) impairs Mg2+ binding as measured by intrinsic tryptophan fluorescence (Figure 4A), and abrogates Mg2+-stimulated active site conformational changes detected by trypsin and chymotrypsin sensitivity of the Tdp2 metamorphic loop (Figure 3D).	RESULTS
275	279	loop	structure_element	In support of this, we also find that hD350N (mD358N) impairs Mg2+ binding as measured by intrinsic tryptophan fluorescence (Figure 4A), and abrogates Mg2+-stimulated active site conformational changes detected by trypsin and chymotrypsin sensitivity of the Tdp2 metamorphic loop (Figure 3D).	RESULTS
0	4	Tdp2	protein	Tdp2 facilitates NHEJ repair of 5′-phosphotyrosine adducted DSBs	RESULTS
32	50	5′-phosphotyrosine	residue_name	Tdp2 facilitates NHEJ repair of 5′-phosphotyrosine adducted DSBs	RESULTS
13	17	Tdp2	protein	Overall, our Tdp2 structure/activity studies reveal a tuned, 5′-detyrosylation DNA end processing activity and it has been demonstrated that Tdp2 could enable repair of Top2 damage by the non-homologous end-joining (NHEJ) pathway.	RESULTS
18	44	structure/activity studies	experimental_method	Overall, our Tdp2 structure/activity studies reveal a tuned, 5′-detyrosylation DNA end processing activity and it has been demonstrated that Tdp2 could enable repair of Top2 damage by the non-homologous end-joining (NHEJ) pathway.	RESULTS
61	78	5′-detyrosylation	ptm	Overall, our Tdp2 structure/activity studies reveal a tuned, 5′-detyrosylation DNA end processing activity and it has been demonstrated that Tdp2 could enable repair of Top2 damage by the non-homologous end-joining (NHEJ) pathway.	RESULTS
79	82	DNA	chemical	Overall, our Tdp2 structure/activity studies reveal a tuned, 5′-detyrosylation DNA end processing activity and it has been demonstrated that Tdp2 could enable repair of Top2 damage by the non-homologous end-joining (NHEJ) pathway.	RESULTS
141	145	Tdp2	protein	Overall, our Tdp2 structure/activity studies reveal a tuned, 5′-detyrosylation DNA end processing activity and it has been demonstrated that Tdp2 could enable repair of Top2 damage by the non-homologous end-joining (NHEJ) pathway.	RESULTS
169	173	Top2	protein_type	Overall, our Tdp2 structure/activity studies reveal a tuned, 5′-detyrosylation DNA end processing activity and it has been demonstrated that Tdp2 could enable repair of Top2 damage by the non-homologous end-joining (NHEJ) pathway.	RESULTS
38	52	5′-tyrosylated	protein_state	Accordingly, we demonstrate here that 5′-tyrosylated ends are sufficient to severely impair an in vitro reconstituted mammalian NHEJ reaction (Figure 7A, lanes 3 and 6), unless supplemented with catalytic quantities of hTdp2FL (Figure 7A, lane 8).	RESULTS
118	127	mammalian	taxonomy_domain	Accordingly, we demonstrate here that 5′-tyrosylated ends are sufficient to severely impair an in vitro reconstituted mammalian NHEJ reaction (Figure 7A, lanes 3 and 6), unless supplemented with catalytic quantities of hTdp2FL (Figure 7A, lane 8).	RESULTS
219	226	hTdp2FL	protein	Accordingly, we demonstrate here that 5′-tyrosylated ends are sufficient to severely impair an in vitro reconstituted mammalian NHEJ reaction (Figure 7A, lanes 3 and 6), unless supplemented with catalytic quantities of hTdp2FL (Figure 7A, lane 8).	RESULTS
15	23	hTdp2cat	structure_element	Interestingly, hTdp2cat is slightly more effective than hTdp2FL in promoting NHEJ of adducted ends, while a catalytically deficient E152Q mutant was inactive in this assay, supporting the notion that Tdp2 catalytic activity is required to support NHEJ of phosphotyrosyl blocked DSBs (Supplementary Figure S7A).	RESULTS
56	63	hTdp2FL	protein	Interestingly, hTdp2cat is slightly more effective than hTdp2FL in promoting NHEJ of adducted ends, while a catalytically deficient E152Q mutant was inactive in this assay, supporting the notion that Tdp2 catalytic activity is required to support NHEJ of phosphotyrosyl blocked DSBs (Supplementary Figure S7A).	RESULTS
108	131	catalytically deficient	protein_state	Interestingly, hTdp2cat is slightly more effective than hTdp2FL in promoting NHEJ of adducted ends, while a catalytically deficient E152Q mutant was inactive in this assay, supporting the notion that Tdp2 catalytic activity is required to support NHEJ of phosphotyrosyl blocked DSBs (Supplementary Figure S7A).	RESULTS
132	137	E152Q	mutant	Interestingly, hTdp2cat is slightly more effective than hTdp2FL in promoting NHEJ of adducted ends, while a catalytically deficient E152Q mutant was inactive in this assay, supporting the notion that Tdp2 catalytic activity is required to support NHEJ of phosphotyrosyl blocked DSBs (Supplementary Figure S7A).	RESULTS
138	144	mutant	protein_state	Interestingly, hTdp2cat is slightly more effective than hTdp2FL in promoting NHEJ of adducted ends, while a catalytically deficient E152Q mutant was inactive in this assay, supporting the notion that Tdp2 catalytic activity is required to support NHEJ of phosphotyrosyl blocked DSBs (Supplementary Figure S7A).	RESULTS
149	157	inactive	protein_state	Interestingly, hTdp2cat is slightly more effective than hTdp2FL in promoting NHEJ of adducted ends, while a catalytically deficient E152Q mutant was inactive in this assay, supporting the notion that Tdp2 catalytic activity is required to support NHEJ of phosphotyrosyl blocked DSBs (Supplementary Figure S7A).	RESULTS
200	204	Tdp2	protein	Interestingly, hTdp2cat is slightly more effective than hTdp2FL in promoting NHEJ of adducted ends, while a catalytically deficient E152Q mutant was inactive in this assay, supporting the notion that Tdp2 catalytic activity is required to support NHEJ of phosphotyrosyl blocked DSBs (Supplementary Figure S7A).	RESULTS
255	269	phosphotyrosyl	ptm	Interestingly, hTdp2cat is slightly more effective than hTdp2FL in promoting NHEJ of adducted ends, while a catalytically deficient E152Q mutant was inactive in this assay, supporting the notion that Tdp2 catalytic activity is required to support NHEJ of phosphotyrosyl blocked DSBs (Supplementary Figure S7A).	RESULTS
48	56	tyrosine	residue_name	We confirmed that efficient joining of the same tyrosine-adducted substrate in cells (Figure 7B) was dependent on both NHEJ (reduced over 10-fold in ligase IV deficient HCT 116 cells; Supplementary Figure S7B), and Tdp2 (reduced 5-fold in Tdp2 deficient MEFs; Figure 7C).	RESULTS
215	219	Tdp2	protein	We confirmed that efficient joining of the same tyrosine-adducted substrate in cells (Figure 7B) was dependent on both NHEJ (reduced over 10-fold in ligase IV deficient HCT 116 cells; Supplementary Figure S7B), and Tdp2 (reduced 5-fold in Tdp2 deficient MEFs; Figure 7C).	RESULTS
239	243	Tdp2	protein	We confirmed that efficient joining of the same tyrosine-adducted substrate in cells (Figure 7B) was dependent on both NHEJ (reduced over 10-fold in ligase IV deficient HCT 116 cells; Supplementary Figure S7B), and Tdp2 (reduced 5-fold in Tdp2 deficient MEFs; Figure 7C).	RESULTS
144	148	Tdp2	protein	Moreover, products with error (i.e. junctions have missing sequence flanking the adducted terminus) are twice as frequent in cells deficient in Tdp2 (Figure 7D).	RESULTS
52	60	tyrosine	residue_name	Therefore, in accord with previous work, joining of tyrosine adducted ends after Tdp2-mediated detyrosylation is both more efficient and more accurate than joining after endonucleolytic excision (e.g. mediated by Artemis or the Mre11/Rad50/Nbs1 complex).	RESULTS
81	85	Tdp2	protein	Therefore, in accord with previous work, joining of tyrosine adducted ends after Tdp2-mediated detyrosylation is both more efficient and more accurate than joining after endonucleolytic excision (e.g. mediated by Artemis or the Mre11/Rad50/Nbs1 complex).	RESULTS
95	109	detyrosylation	ptm	Therefore, in accord with previous work, joining of tyrosine adducted ends after Tdp2-mediated detyrosylation is both more efficient and more accurate than joining after endonucleolytic excision (e.g. mediated by Artemis or the Mre11/Rad50/Nbs1 complex).	RESULTS
213	220	Artemis	protein	Therefore, in accord with previous work, joining of tyrosine adducted ends after Tdp2-mediated detyrosylation is both more efficient and more accurate than joining after endonucleolytic excision (e.g. mediated by Artemis or the Mre11/Rad50/Nbs1 complex).	RESULTS
228	244	Mre11/Rad50/Nbs1	complex_assembly	Therefore, in accord with previous work, joining of tyrosine adducted ends after Tdp2-mediated detyrosylation is both more efficient and more accurate than joining after endonucleolytic excision (e.g. mediated by Artemis or the Mre11/Rad50/Nbs1 complex).	RESULTS
11	15	Tdp2	protein	Effects of Tdp2 active site SNP-encoded mutants on cellular Tdp2 functions. (A) Cy5 labeled substrates with 5′-phosphate termini (Lanes 1–4) or 5′-tyrosylated termini (Lanes 5–9) were incubated with Ku, the NHEJ ligase (XRCC4, ligase IV and XLF; X-L-X) and 1 nM hTdp2FL as indicated (+) for 5 min at 37°C.	FIG
16	27	active site	site	Effects of Tdp2 active site SNP-encoded mutants on cellular Tdp2 functions. (A) Cy5 labeled substrates with 5′-phosphate termini (Lanes 1–4) or 5′-tyrosylated termini (Lanes 5–9) were incubated with Ku, the NHEJ ligase (XRCC4, ligase IV and XLF; X-L-X) and 1 nM hTdp2FL as indicated (+) for 5 min at 37°C.	FIG
60	64	Tdp2	protein	Effects of Tdp2 active site SNP-encoded mutants on cellular Tdp2 functions. (A) Cy5 labeled substrates with 5′-phosphate termini (Lanes 1–4) or 5′-tyrosylated termini (Lanes 5–9) were incubated with Ku, the NHEJ ligase (XRCC4, ligase IV and XLF; X-L-X) and 1 nM hTdp2FL as indicated (+) for 5 min at 37°C.	FIG
80	83	Cy5	chemical	Effects of Tdp2 active site SNP-encoded mutants on cellular Tdp2 functions. (A) Cy5 labeled substrates with 5′-phosphate termini (Lanes 1–4) or 5′-tyrosylated termini (Lanes 5–9) were incubated with Ku, the NHEJ ligase (XRCC4, ligase IV and XLF; X-L-X) and 1 nM hTdp2FL as indicated (+) for 5 min at 37°C.	FIG
108	120	5′-phosphate	chemical	Effects of Tdp2 active site SNP-encoded mutants on cellular Tdp2 functions. (A) Cy5 labeled substrates with 5′-phosphate termini (Lanes 1–4) or 5′-tyrosylated termini (Lanes 5–9) were incubated with Ku, the NHEJ ligase (XRCC4, ligase IV and XLF; X-L-X) and 1 nM hTdp2FL as indicated (+) for 5 min at 37°C.	FIG
144	158	5′-tyrosylated	protein_state	Effects of Tdp2 active site SNP-encoded mutants on cellular Tdp2 functions. (A) Cy5 labeled substrates with 5′-phosphate termini (Lanes 1–4) or 5′-tyrosylated termini (Lanes 5–9) were incubated with Ku, the NHEJ ligase (XRCC4, ligase IV and XLF; X-L-X) and 1 nM hTdp2FL as indicated (+) for 5 min at 37°C.	FIG
199	201	Ku	protein	Effects of Tdp2 active site SNP-encoded mutants on cellular Tdp2 functions. (A) Cy5 labeled substrates with 5′-phosphate termini (Lanes 1–4) or 5′-tyrosylated termini (Lanes 5–9) were incubated with Ku, the NHEJ ligase (XRCC4, ligase IV and XLF; X-L-X) and 1 nM hTdp2FL as indicated (+) for 5 min at 37°C.	FIG
207	218	NHEJ ligase	protein_type	Effects of Tdp2 active site SNP-encoded mutants on cellular Tdp2 functions. (A) Cy5 labeled substrates with 5′-phosphate termini (Lanes 1–4) or 5′-tyrosylated termini (Lanes 5–9) were incubated with Ku, the NHEJ ligase (XRCC4, ligase IV and XLF; X-L-X) and 1 nM hTdp2FL as indicated (+) for 5 min at 37°C.	FIG
220	225	XRCC4	protein	Effects of Tdp2 active site SNP-encoded mutants on cellular Tdp2 functions. (A) Cy5 labeled substrates with 5′-phosphate termini (Lanes 1–4) or 5′-tyrosylated termini (Lanes 5–9) were incubated with Ku, the NHEJ ligase (XRCC4, ligase IV and XLF; X-L-X) and 1 nM hTdp2FL as indicated (+) for 5 min at 37°C.	FIG
227	236	ligase IV	protein	Effects of Tdp2 active site SNP-encoded mutants on cellular Tdp2 functions. (A) Cy5 labeled substrates with 5′-phosphate termini (Lanes 1–4) or 5′-tyrosylated termini (Lanes 5–9) were incubated with Ku, the NHEJ ligase (XRCC4, ligase IV and XLF; X-L-X) and 1 nM hTdp2FL as indicated (+) for 5 min at 37°C.	FIG
241	244	XLF	protein	Effects of Tdp2 active site SNP-encoded mutants on cellular Tdp2 functions. (A) Cy5 labeled substrates with 5′-phosphate termini (Lanes 1–4) or 5′-tyrosylated termini (Lanes 5–9) were incubated with Ku, the NHEJ ligase (XRCC4, ligase IV and XLF; X-L-X) and 1 nM hTdp2FL as indicated (+) for 5 min at 37°C.	FIG
262	269	hTdp2FL	protein	Effects of Tdp2 active site SNP-encoded mutants on cellular Tdp2 functions. (A) Cy5 labeled substrates with 5′-phosphate termini (Lanes 1–4) or 5′-tyrosylated termini (Lanes 5–9) were incubated with Ku, the NHEJ ligase (XRCC4, ligase IV and XLF; X-L-X) and 1 nM hTdp2FL as indicated (+) for 5 min at 37°C.	FIG
49	60	native PAGE	experimental_method	Concatemer ligation products were detected by 5% native PAGE.	FIG
24	51	cellular end joining assays	experimental_method	(B) Workflow diagram of cellular end joining assays.	FIG
0	3	DNA	chemical	DNA substrates with 5′-phosphotyrosine adducts and 4 nucleotide 5′ overhangs were electroporated into cultured mammalian cells.	FIG
20	38	5′-phosphotyrosine	residue_name	DNA substrates with 5′-phosphotyrosine adducts and 4 nucleotide 5′ overhangs were electroporated into cultured mammalian cells.	FIG
111	120	mammalian	taxonomy_domain	DNA substrates with 5′-phosphotyrosine adducts and 4 nucleotide 5′ overhangs were electroporated into cultured mammalian cells.	FIG
11	14	DNA	chemical	After 1 h, DNA was recovered from cells and repair efficiency by qPCR or sequencing as indicated. (C) qPCR assessment of cellular end joining efficiency of the tyrosylated substrate comparing results from wildtype MEF cells to Tdp2−/− cells and Tdp2−/− cells complemented with wildtype or the noted hTDP2FL variants; Joining efficiency shown is the ratio of junctions recovered relative to WT cells.	FIG
65	69	qPCR	experimental_method	After 1 h, DNA was recovered from cells and repair efficiency by qPCR or sequencing as indicated. (C) qPCR assessment of cellular end joining efficiency of the tyrosylated substrate comparing results from wildtype MEF cells to Tdp2−/− cells and Tdp2−/− cells complemented with wildtype or the noted hTDP2FL variants; Joining efficiency shown is the ratio of junctions recovered relative to WT cells.	FIG
73	83	sequencing	experimental_method	After 1 h, DNA was recovered from cells and repair efficiency by qPCR or sequencing as indicated. (C) qPCR assessment of cellular end joining efficiency of the tyrosylated substrate comparing results from wildtype MEF cells to Tdp2−/− cells and Tdp2−/− cells complemented with wildtype or the noted hTDP2FL variants; Joining efficiency shown is the ratio of junctions recovered relative to WT cells.	FIG
102	106	qPCR	experimental_method	After 1 h, DNA was recovered from cells and repair efficiency by qPCR or sequencing as indicated. (C) qPCR assessment of cellular end joining efficiency of the tyrosylated substrate comparing results from wildtype MEF cells to Tdp2−/− cells and Tdp2−/− cells complemented with wildtype or the noted hTDP2FL variants; Joining efficiency shown is the ratio of junctions recovered relative to WT cells.	FIG
160	171	tyrosylated	protein_state	After 1 h, DNA was recovered from cells and repair efficiency by qPCR or sequencing as indicated. (C) qPCR assessment of cellular end joining efficiency of the tyrosylated substrate comparing results from wildtype MEF cells to Tdp2−/− cells and Tdp2−/− cells complemented with wildtype or the noted hTDP2FL variants; Joining efficiency shown is the ratio of junctions recovered relative to WT cells.	FIG
205	213	wildtype	protein_state	After 1 h, DNA was recovered from cells and repair efficiency by qPCR or sequencing as indicated. (C) qPCR assessment of cellular end joining efficiency of the tyrosylated substrate comparing results from wildtype MEF cells to Tdp2−/− cells and Tdp2−/− cells complemented with wildtype or the noted hTDP2FL variants; Joining efficiency shown is the ratio of junctions recovered relative to WT cells.	FIG
227	231	Tdp2	protein	After 1 h, DNA was recovered from cells and repair efficiency by qPCR or sequencing as indicated. (C) qPCR assessment of cellular end joining efficiency of the tyrosylated substrate comparing results from wildtype MEF cells to Tdp2−/− cells and Tdp2−/− cells complemented with wildtype or the noted hTDP2FL variants; Joining efficiency shown is the ratio of junctions recovered relative to WT cells.	FIG
245	249	Tdp2	protein	After 1 h, DNA was recovered from cells and repair efficiency by qPCR or sequencing as indicated. (C) qPCR assessment of cellular end joining efficiency of the tyrosylated substrate comparing results from wildtype MEF cells to Tdp2−/− cells and Tdp2−/− cells complemented with wildtype or the noted hTDP2FL variants; Joining efficiency shown is the ratio of junctions recovered relative to WT cells.	FIG
277	285	wildtype	protein_state	After 1 h, DNA was recovered from cells and repair efficiency by qPCR or sequencing as indicated. (C) qPCR assessment of cellular end joining efficiency of the tyrosylated substrate comparing results from wildtype MEF cells to Tdp2−/− cells and Tdp2−/− cells complemented with wildtype or the noted hTDP2FL variants; Joining efficiency shown is the ratio of junctions recovered relative to WT cells.	FIG
299	306	hTDP2FL	protein	After 1 h, DNA was recovered from cells and repair efficiency by qPCR or sequencing as indicated. (C) qPCR assessment of cellular end joining efficiency of the tyrosylated substrate comparing results from wildtype MEF cells to Tdp2−/− cells and Tdp2−/− cells complemented with wildtype or the noted hTDP2FL variants; Joining efficiency shown is the ratio of junctions recovered relative to WT cells.	FIG
390	392	WT	protein_state	After 1 h, DNA was recovered from cells and repair efficiency by qPCR or sequencing as indicated. (C) qPCR assessment of cellular end joining efficiency of the tyrosylated substrate comparing results from wildtype MEF cells to Tdp2−/− cells and Tdp2−/− cells complemented with wildtype or the noted hTDP2FL variants; Joining efficiency shown is the ratio of junctions recovered relative to WT cells.	FIG
53	80	cellular end-joining assays	experimental_method	Error bars, s.d, n = 3. (D) Junctions recovered from cellular end-joining assays in the noted cell types were characterized by sequencing to assess the end-joining error rate.	FIG
127	137	sequencing	experimental_method	Error bars, s.d, n = 3. (D) Junctions recovered from cellular end-joining assays in the noted cell types were characterized by sequencing to assess the end-joining error rate.	FIG
152	174	end-joining error rate	evidence	Error bars, s.d, n = 3. (D) Junctions recovered from cellular end-joining assays in the noted cell types were characterized by sequencing to assess the end-joining error rate.	FIG
28	53	Clonogenic survival assay	experimental_method	Error bars, s.d, n = 3. (E) Clonogenic survival assay of WT, Tdp2 knockout and complemented MEF cells after treatment with indicated concentrations of etoposide for 3 h; error bars, s.d, n = 3.	FIG
57	59	WT	protein_state	Error bars, s.d, n = 3. (E) Clonogenic survival assay of WT, Tdp2 knockout and complemented MEF cells after treatment with indicated concentrations of etoposide for 3 h; error bars, s.d, n = 3.	FIG
61	65	Tdp2	protein	Error bars, s.d, n = 3. (E) Clonogenic survival assay of WT, Tdp2 knockout and complemented MEF cells after treatment with indicated concentrations of etoposide for 3 h; error bars, s.d, n = 3.	FIG
151	160	etoposide	chemical	Error bars, s.d, n = 3. (E) Clonogenic survival assay of WT, Tdp2 knockout and complemented MEF cells after treatment with indicated concentrations of etoposide for 3 h; error bars, s.d, n = 3.	FIG
32	41	wild-type	protein_state	We next compared the ability of wild-type and mutant hTdp2FL variants to complement Tdp2 deficient mouse embryonic fibroblasts (Supplementary Figure S7C).	RESULTS
46	52	mutant	protein_state	We next compared the ability of wild-type and mutant hTdp2FL variants to complement Tdp2 deficient mouse embryonic fibroblasts (Supplementary Figure S7C).	RESULTS
53	60	hTdp2FL	protein	We next compared the ability of wild-type and mutant hTdp2FL variants to complement Tdp2 deficient mouse embryonic fibroblasts (Supplementary Figure S7C).	RESULTS
84	88	Tdp2	protein	We next compared the ability of wild-type and mutant hTdp2FL variants to complement Tdp2 deficient mouse embryonic fibroblasts (Supplementary Figure S7C).	RESULTS
99	104	mouse	taxonomy_domain	We next compared the ability of wild-type and mutant hTdp2FL variants to complement Tdp2 deficient mouse embryonic fibroblasts (Supplementary Figure S7C).	RESULTS
28	31	DNA	chemical	Joining of extrachromosomal DNA with phosphotyrosine blocked ends, both in terms of efficiency (Figure 7C) and fidelity (Figure 7D), was indistinguishable comparing MEFs from a wild-type mouse, MEFs from a Tdp2-/- mouse overexpressing wild-type human Tdp2, and Tdp2 -/- MEFs overexpressing the I307V variant human Tdp2.	RESULTS
37	52	phosphotyrosine	residue_name	Joining of extrachromosomal DNA with phosphotyrosine blocked ends, both in terms of efficiency (Figure 7C) and fidelity (Figure 7D), was indistinguishable comparing MEFs from a wild-type mouse, MEFs from a Tdp2-/- mouse overexpressing wild-type human Tdp2, and Tdp2 -/- MEFs overexpressing the I307V variant human Tdp2.	RESULTS
177	186	wild-type	protein_state	Joining of extrachromosomal DNA with phosphotyrosine blocked ends, both in terms of efficiency (Figure 7C) and fidelity (Figure 7D), was indistinguishable comparing MEFs from a wild-type mouse, MEFs from a Tdp2-/- mouse overexpressing wild-type human Tdp2, and Tdp2 -/- MEFs overexpressing the I307V variant human Tdp2.	RESULTS
187	192	mouse	taxonomy_domain	Joining of extrachromosomal DNA with phosphotyrosine blocked ends, both in terms of efficiency (Figure 7C) and fidelity (Figure 7D), was indistinguishable comparing MEFs from a wild-type mouse, MEFs from a Tdp2-/- mouse overexpressing wild-type human Tdp2, and Tdp2 -/- MEFs overexpressing the I307V variant human Tdp2.	RESULTS
206	210	Tdp2	protein	Joining of extrachromosomal DNA with phosphotyrosine blocked ends, both in terms of efficiency (Figure 7C) and fidelity (Figure 7D), was indistinguishable comparing MEFs from a wild-type mouse, MEFs from a Tdp2-/- mouse overexpressing wild-type human Tdp2, and Tdp2 -/- MEFs overexpressing the I307V variant human Tdp2.	RESULTS
214	219	mouse	taxonomy_domain	Joining of extrachromosomal DNA with phosphotyrosine blocked ends, both in terms of efficiency (Figure 7C) and fidelity (Figure 7D), was indistinguishable comparing MEFs from a wild-type mouse, MEFs from a Tdp2-/- mouse overexpressing wild-type human Tdp2, and Tdp2 -/- MEFs overexpressing the I307V variant human Tdp2.	RESULTS
235	244	wild-type	protein_state	Joining of extrachromosomal DNA with phosphotyrosine blocked ends, both in terms of efficiency (Figure 7C) and fidelity (Figure 7D), was indistinguishable comparing MEFs from a wild-type mouse, MEFs from a Tdp2-/- mouse overexpressing wild-type human Tdp2, and Tdp2 -/- MEFs overexpressing the I307V variant human Tdp2.	RESULTS
245	250	human	species	Joining of extrachromosomal DNA with phosphotyrosine blocked ends, both in terms of efficiency (Figure 7C) and fidelity (Figure 7D), was indistinguishable comparing MEFs from a wild-type mouse, MEFs from a Tdp2-/- mouse overexpressing wild-type human Tdp2, and Tdp2 -/- MEFs overexpressing the I307V variant human Tdp2.	RESULTS
251	255	Tdp2	protein	Joining of extrachromosomal DNA with phosphotyrosine blocked ends, both in terms of efficiency (Figure 7C) and fidelity (Figure 7D), was indistinguishable comparing MEFs from a wild-type mouse, MEFs from a Tdp2-/- mouse overexpressing wild-type human Tdp2, and Tdp2 -/- MEFs overexpressing the I307V variant human Tdp2.	RESULTS
261	265	Tdp2	protein	Joining of extrachromosomal DNA with phosphotyrosine blocked ends, both in terms of efficiency (Figure 7C) and fidelity (Figure 7D), was indistinguishable comparing MEFs from a wild-type mouse, MEFs from a Tdp2-/- mouse overexpressing wild-type human Tdp2, and Tdp2 -/- MEFs overexpressing the I307V variant human Tdp2.	RESULTS
294	299	I307V	mutant	Joining of extrachromosomal DNA with phosphotyrosine blocked ends, both in terms of efficiency (Figure 7C) and fidelity (Figure 7D), was indistinguishable comparing MEFs from a wild-type mouse, MEFs from a Tdp2-/- mouse overexpressing wild-type human Tdp2, and Tdp2 -/- MEFs overexpressing the I307V variant human Tdp2.	RESULTS
300	307	variant	protein_state	Joining of extrachromosomal DNA with phosphotyrosine blocked ends, both in terms of efficiency (Figure 7C) and fidelity (Figure 7D), was indistinguishable comparing MEFs from a wild-type mouse, MEFs from a Tdp2-/- mouse overexpressing wild-type human Tdp2, and Tdp2 -/- MEFs overexpressing the I307V variant human Tdp2.	RESULTS
308	313	human	species	Joining of extrachromosomal DNA with phosphotyrosine blocked ends, both in terms of efficiency (Figure 7C) and fidelity (Figure 7D), was indistinguishable comparing MEFs from a wild-type mouse, MEFs from a Tdp2-/- mouse overexpressing wild-type human Tdp2, and Tdp2 -/- MEFs overexpressing the I307V variant human Tdp2.	RESULTS
314	318	Tdp2	protein	Joining of extrachromosomal DNA with phosphotyrosine blocked ends, both in terms of efficiency (Figure 7C) and fidelity (Figure 7D), was indistinguishable comparing MEFs from a wild-type mouse, MEFs from a Tdp2-/- mouse overexpressing wild-type human Tdp2, and Tdp2 -/- MEFs overexpressing the I307V variant human Tdp2.	RESULTS
78	82	Tdp2	protein	In contrast, joining of 5′ phosphotyrosine-blocked ends was reduced 5-fold in Tdp2-/- MEFs, and an equivalent defect was observed in Tdp2-/- MEFs overexpressing Tdp2 D350N.	RESULTS
133	137	Tdp2	protein	In contrast, joining of 5′ phosphotyrosine-blocked ends was reduced 5-fold in Tdp2-/- MEFs, and an equivalent defect was observed in Tdp2-/- MEFs overexpressing Tdp2 D350N.	RESULTS
161	165	Tdp2	protein	In contrast, joining of 5′ phosphotyrosine-blocked ends was reduced 5-fold in Tdp2-/- MEFs, and an equivalent defect was observed in Tdp2-/- MEFs overexpressing Tdp2 D350N.	RESULTS
166	171	D350N	mutant	In contrast, joining of 5′ phosphotyrosine-blocked ends was reduced 5-fold in Tdp2-/- MEFs, and an equivalent defect was observed in Tdp2-/- MEFs overexpressing Tdp2 D350N.	RESULTS
71	75	Tdp2	protein	Moreover, the frequency of inaccurate repair was 2-fold higher in both Tdp2 deficient cells and Tdp2 deficient cells overexpressing D350N, relative to cells expressing wild type Tdp2 or hTdp2 I307V (Figure 7D).	RESULTS
96	100	Tdp2	protein	Moreover, the frequency of inaccurate repair was 2-fold higher in both Tdp2 deficient cells and Tdp2 deficient cells overexpressing D350N, relative to cells expressing wild type Tdp2 or hTdp2 I307V (Figure 7D).	RESULTS
132	137	D350N	mutant	Moreover, the frequency of inaccurate repair was 2-fold higher in both Tdp2 deficient cells and Tdp2 deficient cells overexpressing D350N, relative to cells expressing wild type Tdp2 or hTdp2 I307V (Figure 7D).	RESULTS
168	177	wild type	protein_state	Moreover, the frequency of inaccurate repair was 2-fold higher in both Tdp2 deficient cells and Tdp2 deficient cells overexpressing D350N, relative to cells expressing wild type Tdp2 or hTdp2 I307V (Figure 7D).	RESULTS
178	182	Tdp2	protein	Moreover, the frequency of inaccurate repair was 2-fold higher in both Tdp2 deficient cells and Tdp2 deficient cells overexpressing D350N, relative to cells expressing wild type Tdp2 or hTdp2 I307V (Figure 7D).	RESULTS
186	191	hTdp2	protein	Moreover, the frequency of inaccurate repair was 2-fold higher in both Tdp2 deficient cells and Tdp2 deficient cells overexpressing D350N, relative to cells expressing wild type Tdp2 or hTdp2 I307V (Figure 7D).	RESULTS
192	197	I307V	mutant	Moreover, the frequency of inaccurate repair was 2-fold higher in both Tdp2 deficient cells and Tdp2 deficient cells overexpressing D350N, relative to cells expressing wild type Tdp2 or hTdp2 I307V (Figure 7D).	RESULTS
14	23	wild type	protein_state	Expression of wild type or I307V human Tdp2 in Tdp2-/- MEFs was also sufficient to confer levels of resistance to etoposide comparable to the matched wild-type MEF line, while overexpression of human D350N Tdp2 had no apparent complementation activity (Figure 7E).	RESULTS
27	32	I307V	mutant	Expression of wild type or I307V human Tdp2 in Tdp2-/- MEFs was also sufficient to confer levels of resistance to etoposide comparable to the matched wild-type MEF line, while overexpression of human D350N Tdp2 had no apparent complementation activity (Figure 7E).	RESULTS
33	38	human	species	Expression of wild type or I307V human Tdp2 in Tdp2-/- MEFs was also sufficient to confer levels of resistance to etoposide comparable to the matched wild-type MEF line, while overexpression of human D350N Tdp2 had no apparent complementation activity (Figure 7E).	RESULTS
39	43	Tdp2	protein	Expression of wild type or I307V human Tdp2 in Tdp2-/- MEFs was also sufficient to confer levels of resistance to etoposide comparable to the matched wild-type MEF line, while overexpression of human D350N Tdp2 had no apparent complementation activity (Figure 7E).	RESULTS
47	51	Tdp2	protein	Expression of wild type or I307V human Tdp2 in Tdp2-/- MEFs was also sufficient to confer levels of resistance to etoposide comparable to the matched wild-type MEF line, while overexpression of human D350N Tdp2 had no apparent complementation activity (Figure 7E).	RESULTS
114	123	etoposide	chemical	Expression of wild type or I307V human Tdp2 in Tdp2-/- MEFs was also sufficient to confer levels of resistance to etoposide comparable to the matched wild-type MEF line, while overexpression of human D350N Tdp2 had no apparent complementation activity (Figure 7E).	RESULTS
150	159	wild-type	protein_state	Expression of wild type or I307V human Tdp2 in Tdp2-/- MEFs was also sufficient to confer levels of resistance to etoposide comparable to the matched wild-type MEF line, while overexpression of human D350N Tdp2 had no apparent complementation activity (Figure 7E).	RESULTS
176	190	overexpression	experimental_method	Expression of wild type or I307V human Tdp2 in Tdp2-/- MEFs was also sufficient to confer levels of resistance to etoposide comparable to the matched wild-type MEF line, while overexpression of human D350N Tdp2 had no apparent complementation activity (Figure 7E).	RESULTS
194	199	human	species	Expression of wild type or I307V human Tdp2 in Tdp2-/- MEFs was also sufficient to confer levels of resistance to etoposide comparable to the matched wild-type MEF line, while overexpression of human D350N Tdp2 had no apparent complementation activity (Figure 7E).	RESULTS
200	205	D350N	mutant	Expression of wild type or I307V human Tdp2 in Tdp2-/- MEFs was also sufficient to confer levels of resistance to etoposide comparable to the matched wild-type MEF line, while overexpression of human D350N Tdp2 had no apparent complementation activity (Figure 7E).	RESULTS
206	210	Tdp2	protein	Expression of wild type or I307V human Tdp2 in Tdp2-/- MEFs was also sufficient to confer levels of resistance to etoposide comparable to the matched wild-type MEF line, while overexpression of human D350N Tdp2 had no apparent complementation activity (Figure 7E).	RESULTS
9	14	D350N	mutant	The rare D350N variant is thus inactive by all metrics analyzed.	RESULTS
15	22	variant	protein_state	The rare D350N variant is thus inactive by all metrics analyzed.	RESULTS
31	39	inactive	protein_state	The rare D350N variant is thus inactive by all metrics analyzed.	RESULTS
32	37	I307V	mutant	By comparison the more frequent I307V has only mild effects on in vitro activity, and no detectable impact on cellular assays.	RESULTS
0	4	Top2	protein_type	Top2 chemotherapeutic agents remain frontline treatments, and exposure to the chemical and damaged DNA triggers of Top2-DNA protein crosslink formation are unavoidable.	DISCUSS
99	102	DNA	chemical	Top2 chemotherapeutic agents remain frontline treatments, and exposure to the chemical and damaged DNA triggers of Top2-DNA protein crosslink formation are unavoidable.	DISCUSS
115	119	Top2	protein_type	Top2 chemotherapeutic agents remain frontline treatments, and exposure to the chemical and damaged DNA triggers of Top2-DNA protein crosslink formation are unavoidable.	DISCUSS
120	123	DNA	chemical	Top2 chemotherapeutic agents remain frontline treatments, and exposure to the chemical and damaged DNA triggers of Top2-DNA protein crosslink formation are unavoidable.	DISCUSS
42	45	DNA	chemical	Understanding how cells cope with complex DNA breaks bearing topoisomerase–DNA protein crosslinks is key to deciphering individual responses to chemotherapeutic outcomes and genotoxic agents that poison Top2.	DISCUSS
75	78	DNA	chemical	Understanding how cells cope with complex DNA breaks bearing topoisomerase–DNA protein crosslinks is key to deciphering individual responses to chemotherapeutic outcomes and genotoxic agents that poison Top2.	DISCUSS
203	207	Top2	protein_type	Understanding how cells cope with complex DNA breaks bearing topoisomerase–DNA protein crosslinks is key to deciphering individual responses to chemotherapeutic outcomes and genotoxic agents that poison Top2.	DISCUSS
14	25	mutagenesis	experimental_method	Together with mutagenesis and functional assays, our new Tdp2 structures in the absence of ligands and in complex with DNA damage reveal four novel facets of Tdp2 DNA-protein conjugate processing: (i) The Tdp2 active site is well-suited for accommodating a variety of DNA structures including abasic and bulky alkylated DNA lesions that trigger Top2 poisoning, (ii) High-resolution structural analysis coupled with mutational studies and QM/MM molecular modeling of the Tdp2 reaction coordinate support a single metal-ion mechanism for the diverse clade of EEP domain catalyzed phosphoryl hydrolase reactions, (iii) The Tdp2 active site is conformationally plastic, and undergoes intricate rearrangements upon DNA and Mg2+ cofactor binding and (iv) Naturally occurring Tdp2 variants undermine Tdp2 active site chemistry, cellular and biochemical activities.	DISCUSS
30	47	functional assays	experimental_method	Together with mutagenesis and functional assays, our new Tdp2 structures in the absence of ligands and in complex with DNA damage reveal four novel facets of Tdp2 DNA-protein conjugate processing: (i) The Tdp2 active site is well-suited for accommodating a variety of DNA structures including abasic and bulky alkylated DNA lesions that trigger Top2 poisoning, (ii) High-resolution structural analysis coupled with mutational studies and QM/MM molecular modeling of the Tdp2 reaction coordinate support a single metal-ion mechanism for the diverse clade of EEP domain catalyzed phosphoryl hydrolase reactions, (iii) The Tdp2 active site is conformationally plastic, and undergoes intricate rearrangements upon DNA and Mg2+ cofactor binding and (iv) Naturally occurring Tdp2 variants undermine Tdp2 active site chemistry, cellular and biochemical activities.	DISCUSS
57	61	Tdp2	protein	Together with mutagenesis and functional assays, our new Tdp2 structures in the absence of ligands and in complex with DNA damage reveal four novel facets of Tdp2 DNA-protein conjugate processing: (i) The Tdp2 active site is well-suited for accommodating a variety of DNA structures including abasic and bulky alkylated DNA lesions that trigger Top2 poisoning, (ii) High-resolution structural analysis coupled with mutational studies and QM/MM molecular modeling of the Tdp2 reaction coordinate support a single metal-ion mechanism for the diverse clade of EEP domain catalyzed phosphoryl hydrolase reactions, (iii) The Tdp2 active site is conformationally plastic, and undergoes intricate rearrangements upon DNA and Mg2+ cofactor binding and (iv) Naturally occurring Tdp2 variants undermine Tdp2 active site chemistry, cellular and biochemical activities.	DISCUSS
62	72	structures	evidence	Together with mutagenesis and functional assays, our new Tdp2 structures in the absence of ligands and in complex with DNA damage reveal four novel facets of Tdp2 DNA-protein conjugate processing: (i) The Tdp2 active site is well-suited for accommodating a variety of DNA structures including abasic and bulky alkylated DNA lesions that trigger Top2 poisoning, (ii) High-resolution structural analysis coupled with mutational studies and QM/MM molecular modeling of the Tdp2 reaction coordinate support a single metal-ion mechanism for the diverse clade of EEP domain catalyzed phosphoryl hydrolase reactions, (iii) The Tdp2 active site is conformationally plastic, and undergoes intricate rearrangements upon DNA and Mg2+ cofactor binding and (iv) Naturally occurring Tdp2 variants undermine Tdp2 active site chemistry, cellular and biochemical activities.	DISCUSS
73	90	in the absence of	protein_state	Together with mutagenesis and functional assays, our new Tdp2 structures in the absence of ligands and in complex with DNA damage reveal four novel facets of Tdp2 DNA-protein conjugate processing: (i) The Tdp2 active site is well-suited for accommodating a variety of DNA structures including abasic and bulky alkylated DNA lesions that trigger Top2 poisoning, (ii) High-resolution structural analysis coupled with mutational studies and QM/MM molecular modeling of the Tdp2 reaction coordinate support a single metal-ion mechanism for the diverse clade of EEP domain catalyzed phosphoryl hydrolase reactions, (iii) The Tdp2 active site is conformationally plastic, and undergoes intricate rearrangements upon DNA and Mg2+ cofactor binding and (iv) Naturally occurring Tdp2 variants undermine Tdp2 active site chemistry, cellular and biochemical activities.	DISCUSS
91	98	ligands	chemical	Together with mutagenesis and functional assays, our new Tdp2 structures in the absence of ligands and in complex with DNA damage reveal four novel facets of Tdp2 DNA-protein conjugate processing: (i) The Tdp2 active site is well-suited for accommodating a variety of DNA structures including abasic and bulky alkylated DNA lesions that trigger Top2 poisoning, (ii) High-resolution structural analysis coupled with mutational studies and QM/MM molecular modeling of the Tdp2 reaction coordinate support a single metal-ion mechanism for the diverse clade of EEP domain catalyzed phosphoryl hydrolase reactions, (iii) The Tdp2 active site is conformationally plastic, and undergoes intricate rearrangements upon DNA and Mg2+ cofactor binding and (iv) Naturally occurring Tdp2 variants undermine Tdp2 active site chemistry, cellular and biochemical activities.	DISCUSS
103	118	in complex with	protein_state	Together with mutagenesis and functional assays, our new Tdp2 structures in the absence of ligands and in complex with DNA damage reveal four novel facets of Tdp2 DNA-protein conjugate processing: (i) The Tdp2 active site is well-suited for accommodating a variety of DNA structures including abasic and bulky alkylated DNA lesions that trigger Top2 poisoning, (ii) High-resolution structural analysis coupled with mutational studies and QM/MM molecular modeling of the Tdp2 reaction coordinate support a single metal-ion mechanism for the diverse clade of EEP domain catalyzed phosphoryl hydrolase reactions, (iii) The Tdp2 active site is conformationally plastic, and undergoes intricate rearrangements upon DNA and Mg2+ cofactor binding and (iv) Naturally occurring Tdp2 variants undermine Tdp2 active site chemistry, cellular and biochemical activities.	DISCUSS
119	122	DNA	chemical	Together with mutagenesis and functional assays, our new Tdp2 structures in the absence of ligands and in complex with DNA damage reveal four novel facets of Tdp2 DNA-protein conjugate processing: (i) The Tdp2 active site is well-suited for accommodating a variety of DNA structures including abasic and bulky alkylated DNA lesions that trigger Top2 poisoning, (ii) High-resolution structural analysis coupled with mutational studies and QM/MM molecular modeling of the Tdp2 reaction coordinate support a single metal-ion mechanism for the diverse clade of EEP domain catalyzed phosphoryl hydrolase reactions, (iii) The Tdp2 active site is conformationally plastic, and undergoes intricate rearrangements upon DNA and Mg2+ cofactor binding and (iv) Naturally occurring Tdp2 variants undermine Tdp2 active site chemistry, cellular and biochemical activities.	DISCUSS
158	162	Tdp2	protein	Together with mutagenesis and functional assays, our new Tdp2 structures in the absence of ligands and in complex with DNA damage reveal four novel facets of Tdp2 DNA-protein conjugate processing: (i) The Tdp2 active site is well-suited for accommodating a variety of DNA structures including abasic and bulky alkylated DNA lesions that trigger Top2 poisoning, (ii) High-resolution structural analysis coupled with mutational studies and QM/MM molecular modeling of the Tdp2 reaction coordinate support a single metal-ion mechanism for the diverse clade of EEP domain catalyzed phosphoryl hydrolase reactions, (iii) The Tdp2 active site is conformationally plastic, and undergoes intricate rearrangements upon DNA and Mg2+ cofactor binding and (iv) Naturally occurring Tdp2 variants undermine Tdp2 active site chemistry, cellular and biochemical activities.	DISCUSS
163	166	DNA	chemical	Together with mutagenesis and functional assays, our new Tdp2 structures in the absence of ligands and in complex with DNA damage reveal four novel facets of Tdp2 DNA-protein conjugate processing: (i) The Tdp2 active site is well-suited for accommodating a variety of DNA structures including abasic and bulky alkylated DNA lesions that trigger Top2 poisoning, (ii) High-resolution structural analysis coupled with mutational studies and QM/MM molecular modeling of the Tdp2 reaction coordinate support a single metal-ion mechanism for the diverse clade of EEP domain catalyzed phosphoryl hydrolase reactions, (iii) The Tdp2 active site is conformationally plastic, and undergoes intricate rearrangements upon DNA and Mg2+ cofactor binding and (iv) Naturally occurring Tdp2 variants undermine Tdp2 active site chemistry, cellular and biochemical activities.	DISCUSS
205	209	Tdp2	protein	Together with mutagenesis and functional assays, our new Tdp2 structures in the absence of ligands and in complex with DNA damage reveal four novel facets of Tdp2 DNA-protein conjugate processing: (i) The Tdp2 active site is well-suited for accommodating a variety of DNA structures including abasic and bulky alkylated DNA lesions that trigger Top2 poisoning, (ii) High-resolution structural analysis coupled with mutational studies and QM/MM molecular modeling of the Tdp2 reaction coordinate support a single metal-ion mechanism for the diverse clade of EEP domain catalyzed phosphoryl hydrolase reactions, (iii) The Tdp2 active site is conformationally plastic, and undergoes intricate rearrangements upon DNA and Mg2+ cofactor binding and (iv) Naturally occurring Tdp2 variants undermine Tdp2 active site chemistry, cellular and biochemical activities.	DISCUSS
210	221	active site	site	Together with mutagenesis and functional assays, our new Tdp2 structures in the absence of ligands and in complex with DNA damage reveal four novel facets of Tdp2 DNA-protein conjugate processing: (i) The Tdp2 active site is well-suited for accommodating a variety of DNA structures including abasic and bulky alkylated DNA lesions that trigger Top2 poisoning, (ii) High-resolution structural analysis coupled with mutational studies and QM/MM molecular modeling of the Tdp2 reaction coordinate support a single metal-ion mechanism for the diverse clade of EEP domain catalyzed phosphoryl hydrolase reactions, (iii) The Tdp2 active site is conformationally plastic, and undergoes intricate rearrangements upon DNA and Mg2+ cofactor binding and (iv) Naturally occurring Tdp2 variants undermine Tdp2 active site chemistry, cellular and biochemical activities.	DISCUSS
268	271	DNA	chemical	Together with mutagenesis and functional assays, our new Tdp2 structures in the absence of ligands and in complex with DNA damage reveal four novel facets of Tdp2 DNA-protein conjugate processing: (i) The Tdp2 active site is well-suited for accommodating a variety of DNA structures including abasic and bulky alkylated DNA lesions that trigger Top2 poisoning, (ii) High-resolution structural analysis coupled with mutational studies and QM/MM molecular modeling of the Tdp2 reaction coordinate support a single metal-ion mechanism for the diverse clade of EEP domain catalyzed phosphoryl hydrolase reactions, (iii) The Tdp2 active site is conformationally plastic, and undergoes intricate rearrangements upon DNA and Mg2+ cofactor binding and (iv) Naturally occurring Tdp2 variants undermine Tdp2 active site chemistry, cellular and biochemical activities.	DISCUSS
320	323	DNA	chemical	Together with mutagenesis and functional assays, our new Tdp2 structures in the absence of ligands and in complex with DNA damage reveal four novel facets of Tdp2 DNA-protein conjugate processing: (i) The Tdp2 active site is well-suited for accommodating a variety of DNA structures including abasic and bulky alkylated DNA lesions that trigger Top2 poisoning, (ii) High-resolution structural analysis coupled with mutational studies and QM/MM molecular modeling of the Tdp2 reaction coordinate support a single metal-ion mechanism for the diverse clade of EEP domain catalyzed phosphoryl hydrolase reactions, (iii) The Tdp2 active site is conformationally plastic, and undergoes intricate rearrangements upon DNA and Mg2+ cofactor binding and (iv) Naturally occurring Tdp2 variants undermine Tdp2 active site chemistry, cellular and biochemical activities.	DISCUSS
345	349	Top2	protein_type	Together with mutagenesis and functional assays, our new Tdp2 structures in the absence of ligands and in complex with DNA damage reveal four novel facets of Tdp2 DNA-protein conjugate processing: (i) The Tdp2 active site is well-suited for accommodating a variety of DNA structures including abasic and bulky alkylated DNA lesions that trigger Top2 poisoning, (ii) High-resolution structural analysis coupled with mutational studies and QM/MM molecular modeling of the Tdp2 reaction coordinate support a single metal-ion mechanism for the diverse clade of EEP domain catalyzed phosphoryl hydrolase reactions, (iii) The Tdp2 active site is conformationally plastic, and undergoes intricate rearrangements upon DNA and Mg2+ cofactor binding and (iv) Naturally occurring Tdp2 variants undermine Tdp2 active site chemistry, cellular and biochemical activities.	DISCUSS
382	401	structural analysis	experimental_method	Together with mutagenesis and functional assays, our new Tdp2 structures in the absence of ligands and in complex with DNA damage reveal four novel facets of Tdp2 DNA-protein conjugate processing: (i) The Tdp2 active site is well-suited for accommodating a variety of DNA structures including abasic and bulky alkylated DNA lesions that trigger Top2 poisoning, (ii) High-resolution structural analysis coupled with mutational studies and QM/MM molecular modeling of the Tdp2 reaction coordinate support a single metal-ion mechanism for the diverse clade of EEP domain catalyzed phosphoryl hydrolase reactions, (iii) The Tdp2 active site is conformationally plastic, and undergoes intricate rearrangements upon DNA and Mg2+ cofactor binding and (iv) Naturally occurring Tdp2 variants undermine Tdp2 active site chemistry, cellular and biochemical activities.	DISCUSS
415	433	mutational studies	experimental_method	Together with mutagenesis and functional assays, our new Tdp2 structures in the absence of ligands and in complex with DNA damage reveal four novel facets of Tdp2 DNA-protein conjugate processing: (i) The Tdp2 active site is well-suited for accommodating a variety of DNA structures including abasic and bulky alkylated DNA lesions that trigger Top2 poisoning, (ii) High-resolution structural analysis coupled with mutational studies and QM/MM molecular modeling of the Tdp2 reaction coordinate support a single metal-ion mechanism for the diverse clade of EEP domain catalyzed phosphoryl hydrolase reactions, (iii) The Tdp2 active site is conformationally plastic, and undergoes intricate rearrangements upon DNA and Mg2+ cofactor binding and (iv) Naturally occurring Tdp2 variants undermine Tdp2 active site chemistry, cellular and biochemical activities.	DISCUSS
438	462	QM/MM molecular modeling	experimental_method	Together with mutagenesis and functional assays, our new Tdp2 structures in the absence of ligands and in complex with DNA damage reveal four novel facets of Tdp2 DNA-protein conjugate processing: (i) The Tdp2 active site is well-suited for accommodating a variety of DNA structures including abasic and bulky alkylated DNA lesions that trigger Top2 poisoning, (ii) High-resolution structural analysis coupled with mutational studies and QM/MM molecular modeling of the Tdp2 reaction coordinate support a single metal-ion mechanism for the diverse clade of EEP domain catalyzed phosphoryl hydrolase reactions, (iii) The Tdp2 active site is conformationally plastic, and undergoes intricate rearrangements upon DNA and Mg2+ cofactor binding and (iv) Naturally occurring Tdp2 variants undermine Tdp2 active site chemistry, cellular and biochemical activities.	DISCUSS
470	474	Tdp2	protein	Together with mutagenesis and functional assays, our new Tdp2 structures in the absence of ligands and in complex with DNA damage reveal four novel facets of Tdp2 DNA-protein conjugate processing: (i) The Tdp2 active site is well-suited for accommodating a variety of DNA structures including abasic and bulky alkylated DNA lesions that trigger Top2 poisoning, (ii) High-resolution structural analysis coupled with mutational studies and QM/MM molecular modeling of the Tdp2 reaction coordinate support a single metal-ion mechanism for the diverse clade of EEP domain catalyzed phosphoryl hydrolase reactions, (iii) The Tdp2 active site is conformationally plastic, and undergoes intricate rearrangements upon DNA and Mg2+ cofactor binding and (iv) Naturally occurring Tdp2 variants undermine Tdp2 active site chemistry, cellular and biochemical activities.	DISCUSS
557	560	EEP	structure_element	Together with mutagenesis and functional assays, our new Tdp2 structures in the absence of ligands and in complex with DNA damage reveal four novel facets of Tdp2 DNA-protein conjugate processing: (i) The Tdp2 active site is well-suited for accommodating a variety of DNA structures including abasic and bulky alkylated DNA lesions that trigger Top2 poisoning, (ii) High-resolution structural analysis coupled with mutational studies and QM/MM molecular modeling of the Tdp2 reaction coordinate support a single metal-ion mechanism for the diverse clade of EEP domain catalyzed phosphoryl hydrolase reactions, (iii) The Tdp2 active site is conformationally plastic, and undergoes intricate rearrangements upon DNA and Mg2+ cofactor binding and (iv) Naturally occurring Tdp2 variants undermine Tdp2 active site chemistry, cellular and biochemical activities.	DISCUSS
578	598	phosphoryl hydrolase	protein_type	Together with mutagenesis and functional assays, our new Tdp2 structures in the absence of ligands and in complex with DNA damage reveal four novel facets of Tdp2 DNA-protein conjugate processing: (i) The Tdp2 active site is well-suited for accommodating a variety of DNA structures including abasic and bulky alkylated DNA lesions that trigger Top2 poisoning, (ii) High-resolution structural analysis coupled with mutational studies and QM/MM molecular modeling of the Tdp2 reaction coordinate support a single metal-ion mechanism for the diverse clade of EEP domain catalyzed phosphoryl hydrolase reactions, (iii) The Tdp2 active site is conformationally plastic, and undergoes intricate rearrangements upon DNA and Mg2+ cofactor binding and (iv) Naturally occurring Tdp2 variants undermine Tdp2 active site chemistry, cellular and biochemical activities.	DISCUSS
620	624	Tdp2	protein	Together with mutagenesis and functional assays, our new Tdp2 structures in the absence of ligands and in complex with DNA damage reveal four novel facets of Tdp2 DNA-protein conjugate processing: (i) The Tdp2 active site is well-suited for accommodating a variety of DNA structures including abasic and bulky alkylated DNA lesions that trigger Top2 poisoning, (ii) High-resolution structural analysis coupled with mutational studies and QM/MM molecular modeling of the Tdp2 reaction coordinate support a single metal-ion mechanism for the diverse clade of EEP domain catalyzed phosphoryl hydrolase reactions, (iii) The Tdp2 active site is conformationally plastic, and undergoes intricate rearrangements upon DNA and Mg2+ cofactor binding and (iv) Naturally occurring Tdp2 variants undermine Tdp2 active site chemistry, cellular and biochemical activities.	DISCUSS
625	636	active site	site	Together with mutagenesis and functional assays, our new Tdp2 structures in the absence of ligands and in complex with DNA damage reveal four novel facets of Tdp2 DNA-protein conjugate processing: (i) The Tdp2 active site is well-suited for accommodating a variety of DNA structures including abasic and bulky alkylated DNA lesions that trigger Top2 poisoning, (ii) High-resolution structural analysis coupled with mutational studies and QM/MM molecular modeling of the Tdp2 reaction coordinate support a single metal-ion mechanism for the diverse clade of EEP domain catalyzed phosphoryl hydrolase reactions, (iii) The Tdp2 active site is conformationally plastic, and undergoes intricate rearrangements upon DNA and Mg2+ cofactor binding and (iv) Naturally occurring Tdp2 variants undermine Tdp2 active site chemistry, cellular and biochemical activities.	DISCUSS
640	664	conformationally plastic	protein_state	Together with mutagenesis and functional assays, our new Tdp2 structures in the absence of ligands and in complex with DNA damage reveal four novel facets of Tdp2 DNA-protein conjugate processing: (i) The Tdp2 active site is well-suited for accommodating a variety of DNA structures including abasic and bulky alkylated DNA lesions that trigger Top2 poisoning, (ii) High-resolution structural analysis coupled with mutational studies and QM/MM molecular modeling of the Tdp2 reaction coordinate support a single metal-ion mechanism for the diverse clade of EEP domain catalyzed phosphoryl hydrolase reactions, (iii) The Tdp2 active site is conformationally plastic, and undergoes intricate rearrangements upon DNA and Mg2+ cofactor binding and (iv) Naturally occurring Tdp2 variants undermine Tdp2 active site chemistry, cellular and biochemical activities.	DISCUSS
710	713	DNA	chemical	Together with mutagenesis and functional assays, our new Tdp2 structures in the absence of ligands and in complex with DNA damage reveal four novel facets of Tdp2 DNA-protein conjugate processing: (i) The Tdp2 active site is well-suited for accommodating a variety of DNA structures including abasic and bulky alkylated DNA lesions that trigger Top2 poisoning, (ii) High-resolution structural analysis coupled with mutational studies and QM/MM molecular modeling of the Tdp2 reaction coordinate support a single metal-ion mechanism for the diverse clade of EEP domain catalyzed phosphoryl hydrolase reactions, (iii) The Tdp2 active site is conformationally plastic, and undergoes intricate rearrangements upon DNA and Mg2+ cofactor binding and (iv) Naturally occurring Tdp2 variants undermine Tdp2 active site chemistry, cellular and biochemical activities.	DISCUSS
718	722	Mg2+	chemical	Together with mutagenesis and functional assays, our new Tdp2 structures in the absence of ligands and in complex with DNA damage reveal four novel facets of Tdp2 DNA-protein conjugate processing: (i) The Tdp2 active site is well-suited for accommodating a variety of DNA structures including abasic and bulky alkylated DNA lesions that trigger Top2 poisoning, (ii) High-resolution structural analysis coupled with mutational studies and QM/MM molecular modeling of the Tdp2 reaction coordinate support a single metal-ion mechanism for the diverse clade of EEP domain catalyzed phosphoryl hydrolase reactions, (iii) The Tdp2 active site is conformationally plastic, and undergoes intricate rearrangements upon DNA and Mg2+ cofactor binding and (iv) Naturally occurring Tdp2 variants undermine Tdp2 active site chemistry, cellular and biochemical activities.	DISCUSS
769	773	Tdp2	protein	Together with mutagenesis and functional assays, our new Tdp2 structures in the absence of ligands and in complex with DNA damage reveal four novel facets of Tdp2 DNA-protein conjugate processing: (i) The Tdp2 active site is well-suited for accommodating a variety of DNA structures including abasic and bulky alkylated DNA lesions that trigger Top2 poisoning, (ii) High-resolution structural analysis coupled with mutational studies and QM/MM molecular modeling of the Tdp2 reaction coordinate support a single metal-ion mechanism for the diverse clade of EEP domain catalyzed phosphoryl hydrolase reactions, (iii) The Tdp2 active site is conformationally plastic, and undergoes intricate rearrangements upon DNA and Mg2+ cofactor binding and (iv) Naturally occurring Tdp2 variants undermine Tdp2 active site chemistry, cellular and biochemical activities.	DISCUSS
793	797	Tdp2	protein	Together with mutagenesis and functional assays, our new Tdp2 structures in the absence of ligands and in complex with DNA damage reveal four novel facets of Tdp2 DNA-protein conjugate processing: (i) The Tdp2 active site is well-suited for accommodating a variety of DNA structures including abasic and bulky alkylated DNA lesions that trigger Top2 poisoning, (ii) High-resolution structural analysis coupled with mutational studies and QM/MM molecular modeling of the Tdp2 reaction coordinate support a single metal-ion mechanism for the diverse clade of EEP domain catalyzed phosphoryl hydrolase reactions, (iii) The Tdp2 active site is conformationally plastic, and undergoes intricate rearrangements upon DNA and Mg2+ cofactor binding and (iv) Naturally occurring Tdp2 variants undermine Tdp2 active site chemistry, cellular and biochemical activities.	DISCUSS
798	809	active site	site	Together with mutagenesis and functional assays, our new Tdp2 structures in the absence of ligands and in complex with DNA damage reveal four novel facets of Tdp2 DNA-protein conjugate processing: (i) The Tdp2 active site is well-suited for accommodating a variety of DNA structures including abasic and bulky alkylated DNA lesions that trigger Top2 poisoning, (ii) High-resolution structural analysis coupled with mutational studies and QM/MM molecular modeling of the Tdp2 reaction coordinate support a single metal-ion mechanism for the diverse clade of EEP domain catalyzed phosphoryl hydrolase reactions, (iii) The Tdp2 active site is conformationally plastic, and undergoes intricate rearrangements upon DNA and Mg2+ cofactor binding and (iv) Naturally occurring Tdp2 variants undermine Tdp2 active site chemistry, cellular and biochemical activities.	DISCUSS
31	35	Tdp2	protein	This mechanistic dissection of Tdp2 interactions with damaged DNA and metal cofactor provides a detailed molecular understanding of the mechanism of Tdp2 DNA protein crosslink processing.	DISCUSS
62	65	DNA	chemical	This mechanistic dissection of Tdp2 interactions with damaged DNA and metal cofactor provides a detailed molecular understanding of the mechanism of Tdp2 DNA protein crosslink processing.	DISCUSS
149	153	Tdp2	protein	This mechanistic dissection of Tdp2 interactions with damaged DNA and metal cofactor provides a detailed molecular understanding of the mechanism of Tdp2 DNA protein crosslink processing.	DISCUSS
154	157	DNA	chemical	This mechanistic dissection of Tdp2 interactions with damaged DNA and metal cofactor provides a detailed molecular understanding of the mechanism of Tdp2 DNA protein crosslink processing.	DISCUSS
0	4	Tdp2	protein	Tdp2 was originally identified as a protein conferring resistance to both Top1 and Top2 anti-cancer drugs, however it is hypothesized that the predominant natural source of substrates for Tdp2 are likely the potent DNA damage triggers of Top2 poisoning and Top2 DNA protein crosslinks encountered during transcription.	DISCUSS
74	78	Top1	protein_type	Tdp2 was originally identified as a protein conferring resistance to both Top1 and Top2 anti-cancer drugs, however it is hypothesized that the predominant natural source of substrates for Tdp2 are likely the potent DNA damage triggers of Top2 poisoning and Top2 DNA protein crosslinks encountered during transcription.	DISCUSS
83	87	Top2	protein_type	Tdp2 was originally identified as a protein conferring resistance to both Top1 and Top2 anti-cancer drugs, however it is hypothesized that the predominant natural source of substrates for Tdp2 are likely the potent DNA damage triggers of Top2 poisoning and Top2 DNA protein crosslinks encountered during transcription.	DISCUSS
188	192	Tdp2	protein	Tdp2 was originally identified as a protein conferring resistance to both Top1 and Top2 anti-cancer drugs, however it is hypothesized that the predominant natural source of substrates for Tdp2 are likely the potent DNA damage triggers of Top2 poisoning and Top2 DNA protein crosslinks encountered during transcription.	DISCUSS
215	218	DNA	chemical	Tdp2 was originally identified as a protein conferring resistance to both Top1 and Top2 anti-cancer drugs, however it is hypothesized that the predominant natural source of substrates for Tdp2 are likely the potent DNA damage triggers of Top2 poisoning and Top2 DNA protein crosslinks encountered during transcription.	DISCUSS
238	242	Top2	protein_type	Tdp2 was originally identified as a protein conferring resistance to both Top1 and Top2 anti-cancer drugs, however it is hypothesized that the predominant natural source of substrates for Tdp2 are likely the potent DNA damage triggers of Top2 poisoning and Top2 DNA protein crosslinks encountered during transcription.	DISCUSS
257	261	Top2	protein_type	Tdp2 was originally identified as a protein conferring resistance to both Top1 and Top2 anti-cancer drugs, however it is hypothesized that the predominant natural source of substrates for Tdp2 are likely the potent DNA damage triggers of Top2 poisoning and Top2 DNA protein crosslinks encountered during transcription.	DISCUSS
262	265	DNA	chemical	Tdp2 was originally identified as a protein conferring resistance to both Top1 and Top2 anti-cancer drugs, however it is hypothesized that the predominant natural source of substrates for Tdp2 are likely the potent DNA damage triggers of Top2 poisoning and Top2 DNA protein crosslinks encountered during transcription.	DISCUSS
26	29	DNA	chemical	The properties of complex DNA strand breaks bearing Top2-DNA protein crosslinks necessitate that Tdp2 accommodates both damaged nucleic acid as well as the topoisomerase protein in its active site for catalysis.	DISCUSS
52	56	Top2	protein_type	The properties of complex DNA strand breaks bearing Top2-DNA protein crosslinks necessitate that Tdp2 accommodates both damaged nucleic acid as well as the topoisomerase protein in its active site for catalysis.	DISCUSS
57	60	DNA	chemical	The properties of complex DNA strand breaks bearing Top2-DNA protein crosslinks necessitate that Tdp2 accommodates both damaged nucleic acid as well as the topoisomerase protein in its active site for catalysis.	DISCUSS
97	101	Tdp2	protein	The properties of complex DNA strand breaks bearing Top2-DNA protein crosslinks necessitate that Tdp2 accommodates both damaged nucleic acid as well as the topoisomerase protein in its active site for catalysis.	DISCUSS
156	169	topoisomerase	protein_type	The properties of complex DNA strand breaks bearing Top2-DNA protein crosslinks necessitate that Tdp2 accommodates both damaged nucleic acid as well as the topoisomerase protein in its active site for catalysis.	DISCUSS
185	196	active site	site	The properties of complex DNA strand breaks bearing Top2-DNA protein crosslinks necessitate that Tdp2 accommodates both damaged nucleic acid as well as the topoisomerase protein in its active site for catalysis.	DISCUSS
4	8	Tdp2	protein	The Tdp2 substrate interaction groove facilitates DNA-protein conjugate recognition in two important ways.	DISCUSS
9	37	substrate interaction groove	site	The Tdp2 substrate interaction groove facilitates DNA-protein conjugate recognition in two important ways.	DISCUSS
50	53	DNA	chemical	The Tdp2 substrate interaction groove facilitates DNA-protein conjugate recognition in two important ways.	DISCUSS
11	38	nucleic acid binding trench	site	First, the nucleic acid binding trench is assembled by a dynamic β2Hβ DNA damage-binding loop that is capable of recognizing and processing diverse phosphotyrosyl linkages even in the context of bulky adducts such as ϵA. This is achieved by binding of nucleic acid ‘bases out’ by an extended base-stacking hydrophobic wall of the β2Hβ-loop.	DISCUSS
57	64	dynamic	protein_state	First, the nucleic acid binding trench is assembled by a dynamic β2Hβ DNA damage-binding loop that is capable of recognizing and processing diverse phosphotyrosyl linkages even in the context of bulky adducts such as ϵA. This is achieved by binding of nucleic acid ‘bases out’ by an extended base-stacking hydrophobic wall of the β2Hβ-loop.	DISCUSS
65	93	β2Hβ DNA damage-binding loop	structure_element	First, the nucleic acid binding trench is assembled by a dynamic β2Hβ DNA damage-binding loop that is capable of recognizing and processing diverse phosphotyrosyl linkages even in the context of bulky adducts such as ϵA. This is achieved by binding of nucleic acid ‘bases out’ by an extended base-stacking hydrophobic wall of the β2Hβ-loop.	DISCUSS
148	171	phosphotyrosyl linkages	ptm	First, the nucleic acid binding trench is assembled by a dynamic β2Hβ DNA damage-binding loop that is capable of recognizing and processing diverse phosphotyrosyl linkages even in the context of bulky adducts such as ϵA. This is achieved by binding of nucleic acid ‘bases out’ by an extended base-stacking hydrophobic wall of the β2Hβ-loop.	DISCUSS
217	219	ϵA	chemical	First, the nucleic acid binding trench is assembled by a dynamic β2Hβ DNA damage-binding loop that is capable of recognizing and processing diverse phosphotyrosyl linkages even in the context of bulky adducts such as ϵA. This is achieved by binding of nucleic acid ‘bases out’ by an extended base-stacking hydrophobic wall of the β2Hβ-loop.	DISCUSS
292	305	base-stacking	bond_interaction	First, the nucleic acid binding trench is assembled by a dynamic β2Hβ DNA damage-binding loop that is capable of recognizing and processing diverse phosphotyrosyl linkages even in the context of bulky adducts such as ϵA. This is achieved by binding of nucleic acid ‘bases out’ by an extended base-stacking hydrophobic wall of the β2Hβ-loop.	DISCUSS
306	322	hydrophobic wall	site	First, the nucleic acid binding trench is assembled by a dynamic β2Hβ DNA damage-binding loop that is capable of recognizing and processing diverse phosphotyrosyl linkages even in the context of bulky adducts such as ϵA. This is achieved by binding of nucleic acid ‘bases out’ by an extended base-stacking hydrophobic wall of the β2Hβ-loop.	DISCUSS
330	339	β2Hβ-loop	structure_element	First, the nucleic acid binding trench is assembled by a dynamic β2Hβ DNA damage-binding loop that is capable of recognizing and processing diverse phosphotyrosyl linkages even in the context of bulky adducts such as ϵA. This is achieved by binding of nucleic acid ‘bases out’ by an extended base-stacking hydrophobic wall of the β2Hβ-loop.	DISCUSS
14	19	QM/MM	experimental_method	Secondly, our QM/MM analysis further highlights an enzyme–substrate cation–π interaction as an additional key feature of the Tdp2 protein–DNA crosslink binding and reversal.	DISCUSS
68	88	cation–π interaction	bond_interaction	Secondly, our QM/MM analysis further highlights an enzyme–substrate cation–π interaction as an additional key feature of the Tdp2 protein–DNA crosslink binding and reversal.	DISCUSS
125	129	Tdp2	protein	Secondly, our QM/MM analysis further highlights an enzyme–substrate cation–π interaction as an additional key feature of the Tdp2 protein–DNA crosslink binding and reversal.	DISCUSS
138	141	DNA	chemical	Secondly, our QM/MM analysis further highlights an enzyme–substrate cation–π interaction as an additional key feature of the Tdp2 protein–DNA crosslink binding and reversal.	DISCUSS
4	22	strictly conserved	protein_state	The strictly conserved active site Arg216 appears optimally positioned to stabilize a delocalized charge on the phenolate product of the phosphotyrosyl cleavage reaction through molecular orbital overlap and polarization of the leaving group.	DISCUSS
23	34	active site	site	The strictly conserved active site Arg216 appears optimally positioned to stabilize a delocalized charge on the phenolate product of the phosphotyrosyl cleavage reaction through molecular orbital overlap and polarization of the leaving group.	DISCUSS
35	41	Arg216	residue_name_number	The strictly conserved active site Arg216 appears optimally positioned to stabilize a delocalized charge on the phenolate product of the phosphotyrosyl cleavage reaction through molecular orbital overlap and polarization of the leaving group.	DISCUSS
137	151	phosphotyrosyl	ptm	The strictly conserved active site Arg216 appears optimally positioned to stabilize a delocalized charge on the phenolate product of the phosphotyrosyl cleavage reaction through molecular orbital overlap and polarization of the leaving group.	DISCUSS
58	86	substrate cation–π interface	site	To our knowledge, this is the first proposed example of a substrate cation–π interface exploited to promote a phosphoryl-transfer reaction.	DISCUSS
85	89	Tdp2	protein	This unique feature likely provides an additional level of substrate-specificity for Tdp2 by restricting activity to hydrolysis of aromatic adducts characteristic of Top2cc, picornaviral protein–RNA and Hepatitis B Virus (HBV) protein–DNA processing intermediates.	DISCUSS
166	172	Top2cc	complex_assembly	This unique feature likely provides an additional level of substrate-specificity for Tdp2 by restricting activity to hydrolysis of aromatic adducts characteristic of Top2cc, picornaviral protein–RNA and Hepatitis B Virus (HBV) protein–DNA processing intermediates.	DISCUSS
174	186	picornaviral	taxonomy_domain	This unique feature likely provides an additional level of substrate-specificity for Tdp2 by restricting activity to hydrolysis of aromatic adducts characteristic of Top2cc, picornaviral protein–RNA and Hepatitis B Virus (HBV) protein–DNA processing intermediates.	DISCUSS
195	198	RNA	chemical	This unique feature likely provides an additional level of substrate-specificity for Tdp2 by restricting activity to hydrolysis of aromatic adducts characteristic of Top2cc, picornaviral protein–RNA and Hepatitis B Virus (HBV) protein–DNA processing intermediates.	DISCUSS
203	220	Hepatitis B Virus	taxonomy_domain	This unique feature likely provides an additional level of substrate-specificity for Tdp2 by restricting activity to hydrolysis of aromatic adducts characteristic of Top2cc, picornaviral protein–RNA and Hepatitis B Virus (HBV) protein–DNA processing intermediates.	DISCUSS
222	225	HBV	taxonomy_domain	This unique feature likely provides an additional level of substrate-specificity for Tdp2 by restricting activity to hydrolysis of aromatic adducts characteristic of Top2cc, picornaviral protein–RNA and Hepatitis B Virus (HBV) protein–DNA processing intermediates.	DISCUSS
235	238	DNA	chemical	This unique feature likely provides an additional level of substrate-specificity for Tdp2 by restricting activity to hydrolysis of aromatic adducts characteristic of Top2cc, picornaviral protein–RNA and Hepatitis B Virus (HBV) protein–DNA processing intermediates.	DISCUSS
21	24	EEP	structure_element	By comparison, other EEP nucleases such as Ape1 and Ape2 have evolved robust DNA damage specific endonucleolytic and exonucleolytic activities not shared with Tdp2.	DISCUSS
25	34	nucleases	protein_type	By comparison, other EEP nucleases such as Ape1 and Ape2 have evolved robust DNA damage specific endonucleolytic and exonucleolytic activities not shared with Tdp2.	DISCUSS
43	47	Ape1	protein	By comparison, other EEP nucleases such as Ape1 and Ape2 have evolved robust DNA damage specific endonucleolytic and exonucleolytic activities not shared with Tdp2.	DISCUSS
52	56	Ape2	protein	By comparison, other EEP nucleases such as Ape1 and Ape2 have evolved robust DNA damage specific endonucleolytic and exonucleolytic activities not shared with Tdp2.	DISCUSS
77	80	DNA	chemical	By comparison, other EEP nucleases such as Ape1 and Ape2 have evolved robust DNA damage specific endonucleolytic and exonucleolytic activities not shared with Tdp2.	DISCUSS
159	163	Tdp2	protein	By comparison, other EEP nucleases such as Ape1 and Ape2 have evolved robust DNA damage specific endonucleolytic and exonucleolytic activities not shared with Tdp2.	DISCUSS
26	30	Tdp2	protein	The dynamic nature of the Tdp2 active site presents opportunities for enzyme regulation.	DISCUSS
31	42	active site	site	The dynamic nature of the Tdp2 active site presents opportunities for enzyme regulation.	DISCUSS
56	60	Tdp2	protein	However, whether additional protein factors can bind to Tdp2 and modulate assembly/disassembly of the Tdp2 β2Hβ-loop is unknown.	DISCUSS
102	106	Tdp2	protein	However, whether additional protein factors can bind to Tdp2 and modulate assembly/disassembly of the Tdp2 β2Hβ-loop is unknown.	DISCUSS
107	116	β2Hβ-loop	structure_element	However, whether additional protein factors can bind to Tdp2 and modulate assembly/disassembly of the Tdp2 β2Hβ-loop is unknown.	DISCUSS
35	39	Top2	protein_type	We hypothesize that binding of the Top2 protein component of a DNA–protein crosslink and/or other protein-regulated assembly of the Tdp2 active site might also serve to regulate Tdp2 activity to restrict it from misplaced Top2 processing events, such that it cleaves only topologically trapped or poisoned Top2 molecules when needed.	DISCUSS
63	66	DNA	chemical	We hypothesize that binding of the Top2 protein component of a DNA–protein crosslink and/or other protein-regulated assembly of the Tdp2 active site might also serve to regulate Tdp2 activity to restrict it from misplaced Top2 processing events, such that it cleaves only topologically trapped or poisoned Top2 molecules when needed.	DISCUSS
132	136	Tdp2	protein	We hypothesize that binding of the Top2 protein component of a DNA–protein crosslink and/or other protein-regulated assembly of the Tdp2 active site might also serve to regulate Tdp2 activity to restrict it from misplaced Top2 processing events, such that it cleaves only topologically trapped or poisoned Top2 molecules when needed.	DISCUSS
137	148	active site	site	We hypothesize that binding of the Top2 protein component of a DNA–protein crosslink and/or other protein-regulated assembly of the Tdp2 active site might also serve to regulate Tdp2 activity to restrict it from misplaced Top2 processing events, such that it cleaves only topologically trapped or poisoned Top2 molecules when needed.	DISCUSS
178	182	Tdp2	protein	We hypothesize that binding of the Top2 protein component of a DNA–protein crosslink and/or other protein-regulated assembly of the Tdp2 active site might also serve to regulate Tdp2 activity to restrict it from misplaced Top2 processing events, such that it cleaves only topologically trapped or poisoned Top2 molecules when needed.	DISCUSS
222	226	Top2	protein_type	We hypothesize that binding of the Top2 protein component of a DNA–protein crosslink and/or other protein-regulated assembly of the Tdp2 active site might also serve to regulate Tdp2 activity to restrict it from misplaced Top2 processing events, such that it cleaves only topologically trapped or poisoned Top2 molecules when needed.	DISCUSS
306	310	Top2	protein_type	We hypothesize that binding of the Top2 protein component of a DNA–protein crosslink and/or other protein-regulated assembly of the Tdp2 active site might also serve to regulate Tdp2 activity to restrict it from misplaced Top2 processing events, such that it cleaves only topologically trapped or poisoned Top2 molecules when needed.	DISCUSS
29	39	structures	evidence	Furthermore, high-resolution structures of mouse (Figures 3 and 4) and C. elegans Tdp2 show that a single metal ion typifies the Tdp2 active site from worms to man.	DISCUSS
43	48	mouse	taxonomy_domain	Furthermore, high-resolution structures of mouse (Figures 3 and 4) and C. elegans Tdp2 show that a single metal ion typifies the Tdp2 active site from worms to man.	DISCUSS
71	81	C. elegans	species	Furthermore, high-resolution structures of mouse (Figures 3 and 4) and C. elegans Tdp2 show that a single metal ion typifies the Tdp2 active site from worms to man.	DISCUSS
82	86	Tdp2	protein	Furthermore, high-resolution structures of mouse (Figures 3 and 4) and C. elegans Tdp2 show that a single metal ion typifies the Tdp2 active site from worms to man.	DISCUSS
129	133	Tdp2	protein	Furthermore, high-resolution structures of mouse (Figures 3 and 4) and C. elegans Tdp2 show that a single metal ion typifies the Tdp2 active site from worms to man.	DISCUSS
134	145	active site	site	Furthermore, high-resolution structures of mouse (Figures 3 and 4) and C. elegans Tdp2 show that a single metal ion typifies the Tdp2 active site from worms to man.	DISCUSS
151	156	worms	taxonomy_domain	Furthermore, high-resolution structures of mouse (Figures 3 and 4) and C. elegans Tdp2 show that a single metal ion typifies the Tdp2 active site from worms to man.	DISCUSS
160	163	man	taxonomy_domain	Furthermore, high-resolution structures of mouse (Figures 3 and 4) and C. elegans Tdp2 show that a single metal ion typifies the Tdp2 active site from worms to man.	DISCUSS
83	116	intrinsic tryptophan fluorescence	experimental_method	Herein, we report five additional lines of evidence from metal binding detected by intrinsic tryptophan fluorescence, crystallographic analysis of varied metal cofactor complexes, mutagenesis, Ca2+ inhibition studies and QM/MM analysis that all support a feasible single Mg2+ mediated Tdp2 catalytic mechanism.	DISCUSS
118	143	crystallographic analysis	experimental_method	Herein, we report five additional lines of evidence from metal binding detected by intrinsic tryptophan fluorescence, crystallographic analysis of varied metal cofactor complexes, mutagenesis, Ca2+ inhibition studies and QM/MM analysis that all support a feasible single Mg2+ mediated Tdp2 catalytic mechanism.	DISCUSS
180	191	mutagenesis	experimental_method	Herein, we report five additional lines of evidence from metal binding detected by intrinsic tryptophan fluorescence, crystallographic analysis of varied metal cofactor complexes, mutagenesis, Ca2+ inhibition studies and QM/MM analysis that all support a feasible single Mg2+ mediated Tdp2 catalytic mechanism.	DISCUSS
193	216	Ca2+ inhibition studies	experimental_method	Herein, we report five additional lines of evidence from metal binding detected by intrinsic tryptophan fluorescence, crystallographic analysis of varied metal cofactor complexes, mutagenesis, Ca2+ inhibition studies and QM/MM analysis that all support a feasible single Mg2+ mediated Tdp2 catalytic mechanism.	DISCUSS
221	235	QM/MM analysis	experimental_method	Herein, we report five additional lines of evidence from metal binding detected by intrinsic tryptophan fluorescence, crystallographic analysis of varied metal cofactor complexes, mutagenesis, Ca2+ inhibition studies and QM/MM analysis that all support a feasible single Mg2+ mediated Tdp2 catalytic mechanism.	DISCUSS
271	275	Mg2+	chemical	Herein, we report five additional lines of evidence from metal binding detected by intrinsic tryptophan fluorescence, crystallographic analysis of varied metal cofactor complexes, mutagenesis, Ca2+ inhibition studies and QM/MM analysis that all support a feasible single Mg2+ mediated Tdp2 catalytic mechanism.	DISCUSS
285	289	Tdp2	protein	Herein, we report five additional lines of evidence from metal binding detected by intrinsic tryptophan fluorescence, crystallographic analysis of varied metal cofactor complexes, mutagenesis, Ca2+ inhibition studies and QM/MM analysis that all support a feasible single Mg2+ mediated Tdp2 catalytic mechanism.	DISCUSS
0	9	Etoposide	chemical	Etoposide and other Top2 poisons remain front line anti-cancer drugs, and Tdp2 frameshift mutations in the human population confer hypersensitivity to Top2 poisons including etoposide and doxyrubicin.	DISCUSS
20	24	Top2	protein_type	Etoposide and other Top2 poisons remain front line anti-cancer drugs, and Tdp2 frameshift mutations in the human population confer hypersensitivity to Top2 poisons including etoposide and doxyrubicin.	DISCUSS
74	78	Tdp2	protein	Etoposide and other Top2 poisons remain front line anti-cancer drugs, and Tdp2 frameshift mutations in the human population confer hypersensitivity to Top2 poisons including etoposide and doxyrubicin.	DISCUSS
107	112	human	species	Etoposide and other Top2 poisons remain front line anti-cancer drugs, and Tdp2 frameshift mutations in the human population confer hypersensitivity to Top2 poisons including etoposide and doxyrubicin.	DISCUSS
151	155	Top2	protein_type	Etoposide and other Top2 poisons remain front line anti-cancer drugs, and Tdp2 frameshift mutations in the human population confer hypersensitivity to Top2 poisons including etoposide and doxyrubicin.	DISCUSS
174	183	etoposide	chemical	Etoposide and other Top2 poisons remain front line anti-cancer drugs, and Tdp2 frameshift mutations in the human population confer hypersensitivity to Top2 poisons including etoposide and doxyrubicin.	DISCUSS
188	199	doxyrubicin	chemical	Etoposide and other Top2 poisons remain front line anti-cancer drugs, and Tdp2 frameshift mutations in the human population confer hypersensitivity to Top2 poisons including etoposide and doxyrubicin.	DISCUSS
6	10	Tdp2	protein	Given Tdp2 variation in the human population, links to neurological disease and viral pathogenesis, our finding that TDP2 SNPs ablate catalytic activity has probable implications for modulation of cancer chemotherapy, susceptibility to environmentally linked Top2 poisons, and viral infection.	DISCUSS
28	33	human	species	Given Tdp2 variation in the human population, links to neurological disease and viral pathogenesis, our finding that TDP2 SNPs ablate catalytic activity has probable implications for modulation of cancer chemotherapy, susceptibility to environmentally linked Top2 poisons, and viral infection.	DISCUSS
117	121	TDP2	protein	Given Tdp2 variation in the human population, links to neurological disease and viral pathogenesis, our finding that TDP2 SNPs ablate catalytic activity has probable implications for modulation of cancer chemotherapy, susceptibility to environmentally linked Top2 poisons, and viral infection.	DISCUSS
259	263	Top2	protein_type	Given Tdp2 variation in the human population, links to neurological disease and viral pathogenesis, our finding that TDP2 SNPs ablate catalytic activity has probable implications for modulation of cancer chemotherapy, susceptibility to environmentally linked Top2 poisons, and viral infection.	DISCUSS
277	282	viral	taxonomy_domain	Given Tdp2 variation in the human population, links to neurological disease and viral pathogenesis, our finding that TDP2 SNPs ablate catalytic activity has probable implications for modulation of cancer chemotherapy, susceptibility to environmentally linked Top2 poisons, and viral infection.	DISCUSS
8	12	Tdp2	protein	Lastly, Tdp2 inhibitors may synergize or potentiate cytotoxic effects of current anticancer treatments that target Tdp2.	DISCUSS
115	119	Tdp2	protein	Lastly, Tdp2 inhibitors may synergize or potentiate cytotoxic effects of current anticancer treatments that target Tdp2.	DISCUSS
98	102	Tdp2	protein	Thus, we anticipate this atomic-level and mechanistic definition of the molecular determinants of Tdp2 catalysis and conformational changes driven by DNA–protein and protein–protein interactions will foster unique strategies for the development of Tdp2 targeted small molecule interventions.	DISCUSS
150	153	DNA	chemical	Thus, we anticipate this atomic-level and mechanistic definition of the molecular determinants of Tdp2 catalysis and conformational changes driven by DNA–protein and protein–protein interactions will foster unique strategies for the development of Tdp2 targeted small molecule interventions.	DISCUSS
248	252	Tdp2	protein	Thus, we anticipate this atomic-level and mechanistic definition of the molecular determinants of Tdp2 catalysis and conformational changes driven by DNA–protein and protein–protein interactions will foster unique strategies for the development of Tdp2 targeted small molecule interventions.	DISCUSS