# Structural evidence for an in trans base selection mechanism involving Loop1 in Polymerase mu at an NHEJ double-strand break junction

Jerome Loc'H, Christina Gerodimos, Sandrine Rosario, Mustafa Tekpinar, Michael Lieber, Marc Delarue

► To cite this version:

Jerome Loc'H, Christina Gerodimos, Sandrine Rosario, Mustafa Tekpinar, Michael Lieber, et al. Structural evidence for an in trans base selection mechanism involving Loop1 in Polymerase mu at an NHEJ double-strand break junction. Journal of Biological Chemistry, 2019, pp.jbc.RA119.008739. 10.1074/jbc.RA119.008739. pasteur-02170007

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Structural evidence for an *in trans* base selection mechanism involving Loop1 in
Polymerase mu at an NHEJ double-strand break junction

Jérôme Loc'h¹, Christina A. Gerodimos², Sandrine Rosario¹, Mustafa Tekpinar¹,
Michael R. Lieber² and Marc Delarue¹&

¹Unité de Dynamique Structurale des Macromolécules, Institut Pasteur; UMR 3528 du C.N.R.S., 25 rue du Dr Roux, 75015 Paris, France.

²Departments of Pathology, Biochemistry & Molecular Biology, and Molecular Microbiology & Immunology and Department of Biological Sciences, Section of Molecular & Computational Biology, Norris Comprehensive Cancer Center, University of Southern California Keck School of Medicine, Los Angeles, California 90033.

Running title: *Structural model of the base selection mechanism of pol μ*

& To whom correspondence should be addressed:

¹Unité de Dynamique Structurale des Macromolécules, Institut Pasteur;
UMR 3528 du C.N.R.S., 25 rue du Dr Roux, 75015 Paris, France.

Tel +33 1 45 68 86 05.

marc.delarue@pasteur.fr

**Keywords:** Double-strand break DNA repair/ X-ray Crystallography/ DNA bridging/ DNA synapsis/ DNA polymerase polX family/ V(D)J recombination/ non-homologous DNA end joining/ junctional diversity/ Ternary complex/ Pol μ catalytic cycle

ABSTRACT

Eukaryotic DNA polymerase (Pol) X
family members such as Pol µ and terminal
deoxynucleotidyl transferase (TdT) are
important components for the
nonhomologous DNA end-joining (NHEJ)
pathway. TdT participates in a specialized
version of NHEJ, V(D)J recombination. It
has primarily non-templated polymerase
activity, but can take instructions across
strands from the downstream dsDNA, and
both activities are highly dependent on a
structural element called Loop1. However,
it is unclear whether Pol µ follows the same
mechanism because the structure of its
Loop1 is disordered in available structures.
Here, we used a chimeric TdT harboring
Loop1 of Pol µ that recapitulated the
functional properties of Pol µ in ligation
experiments. We solved three crystal

structures of this TdT chimera bound to
several DNA substrates at 1.96-2.55 Å
resolutions, including a full DNA-double
strand break (DSB) synapsis. We then
modeled the full Pol µ sequence in the
context of one these complexes. The atomic
structure of an NHEJ junction with a pol X
construct that mimics Pol µ in a
reconstituted system explained the
distinctive properties of Pol µ compared
with TdT. The structure suggested a
mechanism of base selection relying on
Loop1 and taking instructions via the *in
trans* templating base independently of the
primer strand. We conclude that our atomic-
level structural observations represent a
paradigm shift for the mechanism of base
selection in the polX family of DNA
polymerases.
INTRODUCTION

Two major DNA repair systems can resolve DNA double strand breaks (DSB): homologous recombination (HR) and non-homologous end joining (NHEJ) (1). HR is an accurate process that takes advantage of the presence in the cell of a DNA duplex that is homologous to the DSB site and that will restore faithfully the DNA integrity (2). In contrast, the NHEJ pathway relies only on the two DNA ends at the DSB sites and does not require the presence of homologous DNA. Importantly, NHEJ is usually error-prone, leading to the loss or the addition of few nucleotides (3). HR can occur only in dividing cells during late S and G2 stages, after synthesis of an homologous DNA molecule, whereas the NHEJ pathway can be potentially activated in all phases of the cell cycle and is thought to be the major DSB repair system in higher eukaryotic cells (4).

The NHEJ pathway involves sequential interactions of proteins allowing stabilization, end processing and ligation of the DSB. The eukaryotic NHEJ machinery is composed of the Ku heterodimer (Ku 70/80), DNA-PKcs, Artemis nuclease, Pol λ and/or Pol μ, and the ligase IV-XRCC4-XLF complex, with accessory roles by PAXX and APLF (5, 6). The same machinery participates in a programmed genetic recombination that occurs in developing lymphocytes called V(D)J recombination (7–9). During this process, TdT incorporates random nucleotides at the coding end in order to increase immune repertoire diversity (10, 11). The expression of TdT is limited to primary lymphoid organs, where B- and T-cell maturation occurs, and consequently TdT does not participate in the NHEJ pathway in other cell types (12).

For the last 50 years, TdT has been described as a template-independent polymerase (13, 14). Indeed, it behaves like a nucleotidyltransferase even in the presence of an *in cis* template strand (Figure 1A). However, recent results demonstrate the ability of this enzyme to

carry out templated activity across strands in the presence of a downstream (in trans) DNA duplex with a 3'-protruding end at high DNA:TdT ratios (15). This activity was also described earlier for Pol μ at an equimolar DNA:polX ratio (16), as well as an intrinsic nucleotidyltransferase activity in the presence of transition metal divalent ions (Figure 1A). TdT and Pol μ are two members of the polymerase X family that share high sequence and structure similarity (17, 18) – see Figure S1 for their alignment. From a structural point of view, the main difference between these two polymerases is the sequence of a long loop (Loop1) composed of 20 amino acids (382-401 in TdT) between the β3 and β4 strands (Figure 1B). In all TdT structures, Loop1 adopts a lariat-like conformation (Figure 1C) that prevents the binding of an uninterrupted template DNA strand (19). In contrast, Loop1 is disordered in Pol μ structures obtained in a gap-filling complex and does not interact with the continuous template DNA molecule used for crystallization (20). Thus far, Pol μ could not be crystallized in complex with a true DNA-DSB substrate, despite substantial efforts from several labs, while TdT could not be crystallized in the presence of a one nt-gapped DNA substrate (Figure 1C).

Extensive biochemical experiments were performed on TdT and Pol μ to better understand the difference between their activities. For instance, Pol μ can acquire a template-independent activity by single point mutation or by exchanging the catalytic metal ions from Mg²⁺ to Mn²⁺ (21). Conversely, it is possible to transform TdT to a template-dependent *in cis* polymerase by a single point mutation that destabilizes Loop1 conformation (22), as probed by regular primer extension tests with a primer-template duplex containing a 5'-end overhang on the template strand. Interestingly, the deletion of Loop1 in Pol μ improves both DNA binding and catalytic efficiency in DNA-templated reactions (*in cis*) but inhibits its weak intrinsic template-independent activity (23). Similar
experiments with TdT lead to the same
conclusion: deletion of Loop1 leads to a
drastic decrease of the untemplated activity
correlated with an increase of the *in cis*
template-dependent activity (22).
Furthermore, grafting Loop1 of Pol μ to a
chimeric TdT (Figure 1B) confers to TdT
an *in cis* templated activity (22) (Figure
1A) and vice versa (23). These results
highlight the importance of Loop1 for the
specific activity of both Pol μ and TdT.
However, the role of Loop1 of Pol μ
specifically in a DNA-bridging context is
far from clear.

Interestingly, crystal structures of
TdT in the presence of a full DNA synapsis
could be obtained (15) and showed that
Loop1 is crucial to maintain a tight binding
of TdT across the DNA synapsis (Figure
1C). This raised the question whether
Loop1 has the same role in Pol μ and
possibly uses a similar mechanism of base
selection or not.

Here we present a complete
functional and structural characterization of
the TdT-Loop1-Pol μ chimera (Figure 1B),
hereafter referred to as TdT-μ chimera. We
previously showed that it is a templated
enzyme across a discontinuous template
strand, taking its instructions *in trans* at 1:1
DNA: TdT ratio (15). We now demonstrate
the biological relevance of this Tdt-μ
chimera protein using an *in vitro* NHEJ
ligation assay which shows functional
properties similar to Pol μ. We then solve
and compare its crystal structure in the apo
form to the one of Pol μ (20), as well as in
the gap-filling mode. We also report the
structure of a ternary complex with the
downstream dsDNA (down-dsDNA),
where Loop1 is fully ordered and prevents
the binding of the upstream dsDNA (up-
dsDNA), but actively participates in the
selection of the incoming dNTP in front of
a template base located *in trans*. Related
studies on LigD in prokaryotes show
striking similarities with this mechanism
(24). Finally, we present the structure of a
ternary complex of TdT-μ chimera with a
full DNA-DSB synapsis and an incoming

nucleotide, which could be used as a model
for Pol μ-DNA DSB complex.

RESULTS

The TdT-Loop1 chimera

The first polX chimera was
described in 2006 (23). In this paper, Juarez
and colleagues created both a Pol μ ΔLoop1
mutant and a chimeric construct (Pol
μ–TdT chimera) in which Loop1 was
replaced by the one from TdT.
Electrophoretic Mobility Shift Assay
(EMSA) experiments with Pol μ ΔLoop1
and different DNA substrates suggested that
Loop1 negatively affects the DNA binding
capacity of Pol μ. Furthermore, deletion of
Loop1 in Pol μ produced a 10-fold
improvement of the catalytic efficiency of
*in cis* templated polymerase activity and
abolished the intrinsic template-
independent activity of Pol μ (23).
Functional assays showed a similar
polymerase activity between Pol μ-TdT
chimera and TdT, in the presence of ssDNA
and template/primer substrate.

We performed similar experiments using a TdT-μ chimera (described in **Figure 1B**), obtained by the grafting of Loop1 of Pol μ in a TdT context (22). Note that in the TdT-Pol μ chimera 29 amino acids were changed, containing Loop1 (20 residues). In addition, 4 residues upstream and 5 residues downstream were changed, thereby also including the SD1 region which stands out as “maximally different” between TdT sequences and Pol μ sequences (25). Comparable template-dependent polymerase activity was observed in TdT-μ chimera and Pol μ using an *in cis* DNA substrate (**Figure 1A**), whereas wild-type TdT displays an essentially un-templated polymerase activity in the same conditions (22). More recently, we demonstrated a similar template-dependent activity both in TdT-μ chimera and Pol μ using an *in trans* DNA substrate (**Figure 1A**) (15). Here we describe further functional studies of TdT-μ chimera using an *in vitro* NHEJ ligation assay.
**Ligation of compatible or incompatible 3' overhangs in the presence of full-length wild-type TdT, TdT-μ chimera and Pol μ**

In **Figure 2**, we show that XRCC4:LigaseIV alone is sufficient for ligation of compatible 4-nt 3' overhangs (Lane 3, 53% efficiency), while the addition of Ku 70/80 ensures an even more efficient (>88%) ligation (Lanes 4-7) (**Figure 2A**). This reflects *in vivo* data showing that Ku 70/80 is not essential for ligation of overhangs containing at least 2 bp of microhomology, which can be generated upon hairpin nicking during V(D)J recombination (39). Sequencing data shows that ligation proceeds without nucleotide addition by a polymerase, likely because rapid base pairing of these overhangs occurs faster than template-independent or template-dependent polymerase activity at the DNA ends (**Figure 2B**).

While XRCC4:LigaseIV is sufficient for ligation of compatible overhangs, we find that substantial ligation of *incompatible* 3' overhangs does not occur in the absence of TdT-wt, Pol μ, or TdT-μ chimera (Lanes 12-14) (**Figure 2A**). Sequencing data shows that at least 1 bp of microhomology must become available through either template-dependent or template-independent nucleotide addition before ligation of the ends can occur (**Figure 2C**). In particular, this data shows that full-length TdT adds nucleotides randomly until at least 1 nucleotide is available for base-pairing with the downstream strand. As expected, this reflects the known template-independent activity of TdT-wt. Conversely, Pol μ adds nucleotides mainly template-dependently, although there are four instances where a template-independent addition of 1 nucleotide occurs prior to templated addition, illustrating a small degree of template-independent activity (**Figure 2C**). Importantly, the activity of TdT-μ chimera is much more like that of Pol μ than that of TdT in terms of template-dependence

because most of the nucleotides added are A and thus complementary to the T overhang of the right-hand DNA end. This clearly indicates that Pol μ Loop1 does indeed confer template-dependent activity to TdT-μ chimera across strands, in the context of a DNA synapsis. In addition to the ligated product *sequence*, the ligation *efficiency* also emphasizes that the chimera is more like Pol μ than TdT. Specifically, ligation in reactions with Pol μ and TdT-μ chimera are very similar in efficiency, in contrast to the efficiency for the TdT reactions (**Figure 2A**, compare lanes 12, 13 and 14). Therefore, it is important to note that both the joining efficiency and junctional sequencing support the conclusion that the chimeric protein behaves like Pol μ rather than TdT.

**Overall structure of TdT-μ chimera apoenzyme or in complex with the incoming nucleotide**

Structures of TdT-μ chimera apoenzyme or as a complex with an incoming dideoxynucleotide were solved at 2.20 Å and 1.96 Å resolution, respectively. The overall architecture of the TdT-μ chimera protein is almost identical to the TdT apoenzyme structure (RMSD of 0.517 Å over 336 Cα atoms using 1JMS PDB). The only notable difference is observed in Loop1, localized between strands β3 and β4. In TdT apoenzyme, this loop adopts a lariat-like conformation, with a clear electron density (19), whereas in TdT-μ chimera apoenzyme, 16 amino acids (384-399) out of 20 are missing in the electron density map (**Figure 3**). Therefore, Pol μ's Loop1 (in the context of TdT) appears to be as flexible as reported in the context of Pol μ apo structure or engaged in a gap-filling complex (20, 26).

Interestingly, in the structure of the TdT-μ-chimera dNTP-Mg²⁺ complex, Loop1 becomes mainly visible, with the exception of residues 394-396, and contains a short α-helix. Some crystal contacts were observed that might stabilize this short helix (especially R393), but the rest of Loop1
Structural model of the base selection mechanism of pol μ

structure is free from such contacts and is
sufficient to prevent the binding of an
uninterrupted template strand (Figure 3), as
described in various TdT-dNTP structures
(27). Here D399 and R403 make specific
hydrogen bonds with the nucleobase
(Figure 3), which is stacked between the
conserved positions W450 and R454. We
note that in a bacterial polX from Th.
thermophilus the equivalent of R454,
namely K263, has also been seen to be
essential for strong binding of dNTP-Mg²⁺
(28). The triphosphate moiety of the dNTP
binds at the same place in all other TdT or
Pol μ structures.

D399 and R403 belong to a specific
sequence motif located at the end of Loop1,
called SD1 and first identified in Romain et
al., 2009 (Figure 1B), and they form an
important salt bridge (D399-K403) in TdT
(15), also probed by site-directed
mutagenesis in (40). D399 is conserved in
Loop1 of TdT and Pol μ; its mutation into a
glutamate in Pol μ leads to the degradation
of the primer strand (27), while the mutation
of R403 in Pol μ results in an increased
nucleotidyltransferase activity (21). Here
we mutated the same position in the context
of the Tdt-μ chimera, and tested the activity
of the R403A mutant for its *in trans*
templated activity (Figure S2). We found a
decreased activity with all four substrates,
thereby confirming the important role of
this side chain suggested by the X-ray
structure.

We also observed a rearrangement
in the catalytic site involving the side chain
of the D434. Specifically, in the apo form
and in fact in all known structures of Pol μ
and TdT, this aspartate makes a salt bridge
with R432 (both D434 and R432 residues
have been shown to be essential in TdT by
site-directed mutagenesis, Romain et al.,
unpublished). Here it changes partners from
R432 to R403, from the SD1 motif,
preventing the correct coordination of
Metal A, which is absent in the structure of
the dNTP-Mg²⁺ complex (Figure S3). We
note that the equivalent of D434 in Pol λ is
seen in both conformations in PDB

structure 1XSN and that Metal A is known
to be the last partner to bind in order to
complete the assembly of the catalytic site
in Pol β (29). A similar (but not identical)
mechanism involving a change of partners
in salt bridges occurs in the catalytic site of
Pol β when switching from the open form to
the closed form (30); specifically, D192
switches from interacting with R258 to
Metal B, while R258 changes rotamer to
interact with E295 and Y296 in motif SD2.

In summary, the presence of the
incoming nucleotide participates in the
organization of Pol μ's Loop1, whereas in
TdT Loop1 is intrinsically ordered and
adopts a similar conformation (rmsd=1.95
Å) in the absence or in the presence of an
incoming nucleotide in known structures.

Exchanging Loop1 allows TdT-μ
chimera to bind a DNA substrate in a
gap-filling mode

To check if TdT-μ chimera
reproduces the known behavior of Pol μ in
those cases where structural data are
available, we co-crystallized TdT-μ
chimera in complex with a 1 nt-gapped
DNA duplex substrate and a non-
hydrolysable nucleotide (Figure 4A). In
this non-hydrolysable dNTP, the oxygen
atom between α- and β-phosphate has been
substituted by a carbon atom, in order to
prevent DNA synthesis and to block the
enzyme in a pre-catalytic state. The
structure was solved at 2.35 Å resolution
with two copies of the complex in the
asymmetric unit. The electron density of
each one of the DNA bases is well-defined
and readily allows the building of the DNA
molecules as well as the incoming
nucleotide, which makes Watson-Crick
interactions with the templating base
(Figure S4A). Binding of the uninterrupted
template strand is possible because residues
384 to 401, corresponding to Loop1, are
disordered (Figure 4C). Such a complex
could not be obtained under the same
conditions using wild-type TdT, probably
because of its intrinsically ordered Loop1,
while several similar structures, also
showing a disordered Loop1, have been solved with Polymerase μ (20, 26, 31). The two copies of the same complex in the asymmetric unit have no major difference (RMSD of 0.28 Å over 329 Cα atoms). Their comparison with the corresponding complex with Pol μ shows that the protein structures are very close (RMSD of 1.95 Å over 328 Cα atoms of the protein using 2IHM PDB), as well as the DNA molecules: the RMSD is 1.255 Å over 11 nucleotides for the template strand, 1.30 Å for 6 nucleotides in the upstream primer strand and 1.36 Å over 4 nucleotides for the downstream primer strand (Figure 4D). In both structures Loop1 is disordered and gives way to the DNA template strand. One difference involves the β2-α12 loop that appears more flexible in the TdT-μ chimera gap-filling complex since no electron density is present to build residues 452 and 453 (Figure 4C), while the N-terminal part of α12 helix is slightly distorted and shifted by 3.3 Å. Concerning the nucleobase of the incoming dNTP, its orientation is slightly modified compared to the one seen in the ddCTP complex to make a Watson-Crick base-pair with the templating base, while the R454 side chain swings to allow this rearrangement (Figure 4B).

In the TdT-μ chimera gap-filling structure, we used a 5'-phosphorylated downstream primer because the presence of a phosphate group in this position was described to be important for the binding of the downstream DNA strands in Pol μ (31, 32). Importantly, we also tested the gap-filling activity in vitro for the TdT-μ chimera and found that it essentially reproduces the activity of Pol μ, and not that of TdT (Figure S5).

## Loop1 checks the *in trans* nucleotide selection in the absence of a DNA primer strand

By mixing TdT-μ chimera with a 2-fold excess of the dsDNA and an incoming ddCTP, we obtained crystals that lead to a detailed picture of a possible role for Loop1 at 2.09 Å resolution. Unexpectedly, only the

downstream dsDNA was visible in the electron density (Figure S4B) and Loop1 was ordered and actively involved, through its main chain atoms, in stabilizing the Watson-Crick interactions of the nascent base-pair with an *in trans* instructing base. All residues of Loop1, including the side chains, could be manually built (Figure 5A and 5B) and several secondary structure elements were identified, including two sequential $\beta_{10}$ helices and an α-helix of seven residues (Figure 6A). Notably, no crystal contact is involved in the stabilization of this conformation. The full upstream dsDNA is excluded by Loop1, whereas in TdT's comparable structure Loop1 just prevents the binding of a continuous template strand but not of the primer strand (Figure 7D). The incoming ddCTP, which can access the nucleotide binding site through a dedicated channel formed by the 8-kDa and fingers domains, makes Watson-Crick interactions with the first 3' protruding base of the downstream template strand and nicely fits a cavity created by Loop1 (Figure 7B), whereas the rest of the protruding bases make their way out of the active site through a separate exit channel, encompassed between Loop1 and the thumb domain (Figure 7C).

The incoming ddCTP is positioned by Loop1 Pol μ opposite the most downstream template possible (the last ssDNA/template nucleotide before dsDNA), even when another upstream complementary nucleotide is present (Figures 5A and 7A). This is consistent with previously described studies of template selection by Pol μ (31,32).

We observe a slight distortion in the catalytic site, where the χ2 angle of the catalytic residue D434 (D418 in Pol μ) is rotated by 82° compared to the apo form (Figure S3). This is due to an interaction with the side chain of H381 (H363 in Pol μ), which is also stabilized by stacking interactions with R403. This rotation prevents the correct coordination of metal A in the active site.
Structural model of the base selection mechanism of pol μ

Loop1 drastically changes its conformations and is completely remodeled when compared to the ddCTP binary complex (Figure 7D). It interacts with residues in the fingers domain (L260), the palm domain (Q379, R403 and D434), and the thumb domain (R454, R461, D473, N474 and H475) of the protein (Figure 6B-D). The interactions with the incoming base involve only main chain atoms of Loop1 (Figure 6C) and the templating base interacts with the conserved residue R461, whose mutation into an alanine has a strong deleterious effect in TdT (25). Importantly, all of the interactions of Loop1 in the chimera construct with the rest of the TdT-like structure involve residues that are conserved in Pol μ or subject to a conservative substitution (Figure 6C). To investigate further how this structure would be modified in the context of the full Pol μ sequence, we modeled this complex using homology modeling techniques. This is justified considering the high level of sequence identity (42%) between them.

## Modeling of the full sequence of pol μ in the context of the downstream-dsDNA complex

Both in the TdT-μ chimera x-ray structure and the pol μ homology model the following features were observed.

- The *main chain atoms* of N391, L392 and R393 amino acids stabilize the nascent base-pair formed by the incoming ddCTP and the template base across strands but their side-chain atoms play no apparent role. This is shown in **Figure 7B**, where residues from Loop1 are represented in surface mode and colored in dark blue, playing the role of the absent upstream dsDNA. Clearly, the check is made at the level of the nascent base-pair *volume* and there is no base specificity: all iso-steric base-pairs would be accommodated in the same way in this cavity.

-There is a direct interaction of the base immediately downstream of the templating base with the side chain of R393, which also

interacts both with the side chains of S388 and N391 in Loop1 (Figure 6C).

-The side chains of Q393B and T397 make hydrogen bonds with the DNH motif (D473, N474 and H475), also called SD2 region (22) or SD2 motif (25), or thumb mini-loop motif (16), localized in the β8-β9 loop (Figure 6D). Mutations of this motif in human pol μ (16) resulted in loss of function.

-There is a van der Waals contact between F401 in region SD1 both with W450 and with the SD2 region. F401A mutation human pol μ (16), resulting in total loss of activity.

-Both residues F401 and F405 (SD1) make a sandwich for the side-chain of H381 (N-terminus of Loop1), thereby clipping both ends of Loop1. The mutation of F405 (F387A in human pol μ), as well as in mouse pol μ (F391A), resulted in a total loss of function (16, 25).

-D399 side-chain stabilizes the short N-terminal helix of Loop1. Its mutation in mouse pol μ (D385E) resulted in a total loss of function (25).

Interestingly, two arginine residues (R454 and R458 in TdT, corresponding to K438 and R442 in Pol μ) are close to position S372 (S388 in TdT), localized in the middle of Loop1, which is the main cyclin-dependent kinase (CDK) phosphorylation site in Pol μ during S and G2 phases (33). A reduced activity of Pol μ was observed when this position was mutated into a glutamate residue, mimicking a phosphorylated serine, suggesting a regulatory mechanism to avoid NHEJ activity in dividing cells. The structure therefore suggests how these two arginines would interact with a phosphorylated serine at position S372 and increase the stability of Loop1, thereby preventing the binding of the primer DNA binding and inhibiting the polymerase activity.

It should be noted that a short extra DNA strand forming a triple helix with each dsDNA is present in the electron density map, forming 1A:2T triple bases. This third strand does not interact with the protein,
except with E67, far away from the active
site, but stabilizes packing interactions that
occur between neighboring DNA duplexes
in the crystal. To check if the presence of
this extra strand could induce an artefactual
conformation of the dsDNA in the crystal,
we compared its structure with an earlier
structure of TdT (PDB code 5D46) with a
DSB-DNA synapsis and found that the
RMSD is 1.16 Å over the backbone atoms
of 12 nucleotides (3 atoms per nucleotide:
C4', C1', P) for the downstream dsDNA.
Furthermore, we removed the third strand
and performed energy minimization in
water: the RMSD of DNA atoms of the
TdT-μ chimera was only 0.8 Å, while the
RMSD on C-alpha atoms was 1.1 Å.

We also subjected the pol μ homology
model to energy minimization in water in
the presence of both ddCTP and the dn-
dsDNA. After equilibration the rmsd on
DNA atoms was 0.8 Å and 1.0 Å for the
protein C-alpha atoms. Notably, Loop1
conformation was remarkably stable.

**Flexibility of Loop1 allows Pol μ to**
**interact with a full DNA synapsis**

We also solved the structure of TdT-
μ chimera bound to a full DSB-DNA
substrate (a DNA synapsis) and with an
incoming ddCTP, at 2.55 Å resolution, by
increasing the dsDNA:protein ratio to 4:1
instead of 2:1. This time, both upstream and
downstream dsDNA can be fully built in the
electron density map (Figure S4C). The
TdT-μ chimera DSB-DNA structure is
highly similar to the TdT-wt DSB-DNA
structure (RMSD of 0.504 Å over 336 Ca
atoms with 5D46 PDB). Moreover, ddCTP
is present in the nucleotide binding pocket
and makes Watson-Crick interactions with
the first single base at the 3' protruding end
of the downstream dsDNA molecule
(across strands). Loop1 appears to be
disordered in this structure so that it does
not sterically hinder the binding of the
template strand (Figure 8A and 8B), as
observed in the TdT-μ chimera structure in
a gap-filling mode (Figure 4).

A third DNA strand is present on
each DNA duplex, forming a triple helix
with a 1A:2T stoichiometry (Figure 8A).
As described in the previous section, these
additional strands help to stabilize
interactions in the crystal packing
arrangement, but they do not interact
directly with the protein. They also do not
directly participate in the stabilization of the
DNA synapsis itself, as observed in the
TdT-wt DSB-DNA complex. To check if
the presence of this extra strand could
induce an artefactual conformation of the
DNA in the crystal, we compared its
structure with the known structure of TdT
with a DSB-DNA synapsis—the RMSD is
0.5 Å over 6 nucleotides for the upstream
primer, 5 nucleotides for the downstream
primer strand and 6 backbones for the
downstream template strand (with 3
backbone atoms per nucleotide). We note
that the third DNA strand is not in the same
direction in the downstream and upstream
parts of the synapsis.

In summary, it is possible to
crystallize the TdT-μ chimera construct in
the context of a full DNA-DSB junction, but
in this case Loop1 is lifted up and moved
out of the way of the upstream DNA duplex,
as if it is not needed any more once it has
played its role to select the base in front of
the *in trans* templating base.

DISCUSSION

The studies presented here provide
the first atomic structure for two DNA ends
brought close together by a protein that
recapitulates the properties of a polX DNA
polymerase involved in the NHEJ
machinery, in this case Pol μ, in ligation
experiments.

Comparison of polX activities during
NHEJ in the presence of different 3'
ends

The biochemical ligation tests using
Ku 70/80, XRCC4:LigaseIV and either the
full-length TdT, Pol μ or TdT-μ chimera,
provide useful insights on the role of polX
polymerases in NHEJ (Figure 2). First, TdT
robustly adds nucleotides in a template-
independent manner prior to ligation in the
case of incompatible DNA ends. Yet, TdT
does not add nucleotides when compatible
DNA ends are being joined. This illustrates
that the collision and annealing of the DNA
ends is rapid relative to the encounter of
those ends with the polX polymerase. This
observation confirms and extends previous
work showing that when DNA end
structures are compatible, then new
synthesis is suppressed. This was indeed
apparent in very early work before specific
proteins were identified for NHEJ (34, 35).
More recently, the degree of polX
engagement was shown to be directly
proportional to the extent to which there
was a barrier to direct ligation (due to
sequence overhang incompatibility), both in
vitro and in vivo, for Pol λ (36).

Second, for incompatible DNA
ends, Pol μ usually adds nucleotides that
generate terminal microhomology. But in
~25% of instances in the experiments here
for this configuration, it appears that Pol μ
adds at least 1 nucleotide in a template-
independent manner. This raises the
possibility that the microhomology
nucleotide is also template-independent,
and we are only observing the subset of
events where Pol μ added, by chance, a
nucleotide that provided 1 bp of terminal
microhomology. The remaining nucleotides
could reflect fill-in synthesis by Pol μ in a
template-dependent manner. The clearest
tests of template-dependent vs. template-
independent addition by Pol μ is with
dideoxynucleotides or immobilized DNA
ends (31, 37, 38), and in these tests Pol μ
shows both template-independent and
template-dependent activity. Our *in trans*
structural studies show synthesis across a
discontinuous template by both Pol μ and
TdT-μ chimera (15). All of the
aforementioned biochemical and structural
data is consistent with the original
conception of Pol μ's ability to cross a
discontinuous template (37).

Most importantly for this study, in
the NHEJ biochemical assays using TdT-μ

chimera, the nucleotide additions are much
more like those of Pol μ than of TdT. This
illustrates the importance of Loop1 in the
distinction between TdT and Pol μ, directly
in the context of NHEJ, and validates that
TdT-μ chimera can be used to characterize
the role of Loop1 in Pol μ and the SD1
region at the structural level.

**Loop1 in the context of the pol X family: positioning SD1 and SD2 regions**

The length of Loop1 is one of the
main differences observed among members
of the polymerase X family. This loop is
composed of only 4 and 9 amino acids in
Polymerase β and Polymerase λ,
respectively, whereas Loop1 is made up of
20 amino acids in TdT and 17-21 residues
in Polymerase μ (Figure 5C). All structures
of individual of members of this family
were solved by X-ray crystallography.
Loop1 can be observed in Pol β, Pol λ and
TdT structures, but not in any of the
currently available Pol μ structures (this
loop is too small in Pol β and Pol λ to
interfere with the template DNA path.). At
the sequence level, Loop1 is more
conserved in TdT sequences than in Pol μ
sequences and there seems to be an inverse
correlation between sequence conservation
and flexibility of Loop1. Indeed, Loop1
always adopts the same fixed correlated
conformation in TdT, where the sequence
conservation is high. On the other hand,
Loop1 sequence is more divergent in Pol μ,
resulting in an increased flexibility of this
loop in Pol μ.

Just downstream of Loop1, there is
an important region called SD1 that is
differentially conserved in Pol μ and TdT
(Figure 1B and Figure 5C). Our structures
indicate that Loop1 ordering in the complex
with the dn-dsDNA is responsible for the
new positioning of the SD1 region (located
at the C-terminus of Loop1) with respect to
the SD2 region and the catalytic site
(Figure 6), and this probably explains its
importance for functional aspects of Pol μ.
For Pol λ, Loop1 is too short to play the role
described here. However, Loop3, coming
from the down-dsDNA side, might be able
to play a similar role, as suggested by the
superimposition of the different structures
in a recent review (39). Answering this
question will require additional structural
studies of Pol λ in the context of a true DNA
synapsis, as we have done here.

Sequence of conformational changes
during Pol μ catalytic cycle in the
presence of 3'overhanging DNA ends

Recent studies have provided a
wealth of structural and biochemical
information about the DNA bridging
binding properties of eukaryotic polX
polymerases (15, 40, 45). Nevertheless, the
order of substrate binding as well as the role
of Loop1 for Pol μ activity in the NHEJ
pathway remains a central unknown aspect.
The new structural information revealed
here by using TdT-μ protein may be
organized as follows to explain the function
of Loop1 during NHEJ pathway in the
presence of 3' protruding ends by Pol μ
(Figure 9). First, our data on the binary
complex with dNTP would be compatible
with the idea that Pol μ is always “loaded”
with a dNTP (see below). When a DSB is
detected and stabilized by Ku heterodimer,
Pol μ-dNTP complex would bind
preferentially to the downstream DNA
duplex, due to the presence of a 5'-
phosphate binding pocket. In this process,
Loop1 is rearranged to stabilize Watson-
Crick interactions in the microhomology
base-pairs across strands and excludes the
binding of the up-dsDNA. Subsequently,
Loop1 would be displaced by the up-
dsDNA positioning, driven by base stacking
interactions. Pol μ would then catalyze
nucleotide incorporation to the primer
DNA, allowing bridging between upstream
and downstream dsDNA, followed by
dissociation of the complex. Therefore, the
catalytic cycle contains a separate step that
checks Watson-Crick interactions at the
nascent base pair, independently of the
upstream DNA molecule. This suggests for
the first time a structural basis for the role
of the specific Loop1 of Pol μ that includes

the *selection* of the incoming nucleotide
*before* binding the upstream dsDNA.
This step is actually the major difference
between TdT and Pol μ, because such an
intermediate state was never detected
during extensive crystallization trials at
various wt TdT:DNA ratios. Indeed, Loop1
is always ordered in TdT and structured in
such a way that it excludes the upstream
template strand, but not the upstream primer
strand, even in a templating mode (52). In
doing so, it further signifies the importance
of stacking interactions with the 3' base,
which indeed are known to play a major role
in the nature of sequences added by TdT
(13, 40).

Similarity with the bacterial NHEJ system

Loop1 is mostly ordered in the
binary structure with dNTP-Mg<sup>++</sup>, in the
absence of any primer strand (Figure 9).
Strikingly, the same type of intermediate
structure is also present in bacterial polX
from *T. thermophilus* (41), where it was
suggested that the bacterial polX is always
present in solution as a complex with one of
the four dNTPs. This is important because
phylogenetic studies of the polX family (41)
suggest that eukaryotic polX members
involved in NHEJ have a bacterial origin.
We note that if the polymerase is already
loaded with a dNTP prior to its binding to a
synapsis of two DNA ends, it may
incorporate a nucleotide that does not match
the downstream DNA end, resulting in a
template-independent mode (37). This is a
critical point highlighted by our study.

In bacteria, NHEJ is promoted by
PolDom, a member of the Archaeo-
Eukaryotic Primase (AEP) superfamily
whose folding is different from the polX
family. In the bacterial *M. tuberculosis*
PolDom structure, there is also an
intermediate state that contains the
incoming nucleotide and only the
downstream dsDNA (with no upstream
dsDNA), and also a mobile loop, called
Loop2, that can adopt two conformations
and regulate the binding of a catalytic metal
ion in the polymerase active site (24). The rotation of the side chain of one of the catalytic aspartates that interacts with an arginine belonging to Loop2 leads to an inactive catalytic site (Figure S6). The relevance of such a complex in solution was demonstrated using fluorescence resonance energy transfer (FRET) experiments and EMSA (24). Although Loop1 of Pol µ is neither structurally nor topologically related in any way to Loop2 of PolDom, both loops intervene in stabilizing the catalytic site conformation. Also, both loops are able to promote the complete exclusion of the upstream dsDNA. It was postulated for PolDom that this preliminary step is responsible for nucleotide selection, prior to DNA bridging at the DSB site. The fact that similar observations can be made in the prokaryotic and eukaryotic NHEJ pathways suggests that this mechanism, which dissociates DNA bridging and fidelity, may have been selected twice in evolution. Because the folding topologies of the polymerases involved in this reaction are different, we may speak of *convergent evolution* for the mechanism of base selection by the NHEJ polymerase in bacterial (AEP family) and eukaryotic (polX family) systems.

In conclusion, the set of proposed structures of intermediates in the catalytic cycle of Pol µ described here represents a paradigm shift in the base selection mechanism in the polX family of DNA polymerases. Because of the high quality in the atomic details of this set of structures and of the high sequence identity between Pol µ and the Tdt-µ chimera, we can reliably model Pol µ in the context of the proposed complexes along the catalytic cycle, which might, ultimately, help in the rational design of inhibitors specific to this step of NHEJ and DNA repair, during which the DNA ends are made compatible before ligation.

## EXPERIMENTAL PROCEDURES

### Cloning and protein purification

The catalytic domain of mouse TdT and mouse TdT-µ was expressed and purified using the protocol described in (25). The catalytic domain of human Pol µ was cloned, expressed and purified using the protocol described in (26). The full-length sequences of mouse TdT and mouse Pol µ were cloned into RSFDuet-1 expression vector (Novagen) fused to an N-terminal 14 histidine-tag followed by a cleavage site for Tobacco Etch Virus (TEV) protease. The full-length sequence of TdT-µ chimera contains the Breast cancer susceptibility C terminus (BRCT) domain of mouse pol µ (1-140), the catalytic domain of mouse TdT (141-510) and Loop1 of mouse pol µ (378-406).

In order to keep the original TdT residue numbering everywhere, including after Loop1, the Q394 residue that is an insertion between the two Loop1 sequences is labelled differently (Q393B, **Figure 1B**).

Proteins were expressed in *E. coli* BL21-CodonPlus (DE3)-RIPL strain in LB (Luria Broth) at 20°C for 16 hours after induction by 0.5 mM of isopropyl-D-thiogalactoside. The purification was done using Ni-Nitrilotriacetic acid (NTA) chromatography, followed by overnight TEV cleavage and heparin chromatography (GE Healthcare). All proteins were stored at -20°C in Tris HCl 25 mM pH 7, NaCl 300 mM and 15% glycerol.

Recombinant Ku70/80 and recombinant XRCC4:Ligase IV were expressed and purified as previously described (42, 43). Proteins were expressed using a baculovirus system in High Five cells (Thermo-Fisher Scientific). Ku 70/80 was purified by Ni-NTA affinity, dsDNA (oligo) affinity, and size exclusion chromatography. XRCC4:Ligase IV was purified by Ni-NTA affinity and two-step ion exchange chromatography.

### Oligonucleotides and DNA Substrates
Oligonucleotides used for the NHEJ assay were synthesized by Integrated DNA Technologies, Inc. (San Diego, CA, USA). Oligonucleotides were purified using 12% denaturing Polyacrylamide gel electrophoresis (PAGE) and their concentration was determined by UV-spectroscopy. 5' end radiolabeling of oligonucleotides was performed using [γ-³²P]ATP (3000 Ci/mol) (PerkinElmer Life Sciences) and T4 polynucleotide kinase (New England Biolabs). Unincorporated radioisotope was removed using Sephadex G-25 spin columns (Epoch Life Science). Duplex DNA substrates were created by adding a 20% excess of unlabeled oligonucleotide to the radiolabeled complementary strand. DNA substrates were heated at 95°C for 5 min and cooled at room temperature for 3 h, then at 4°C overnight. Sequences of oligonucleotides used in this study are as follows: CG07, 5'-C*G*T* T*AA GTA TCT GCA TCT TAC TTG ATG GAG GAT CCT GTC ACG TGC TAG ACT ACT GGT CAA GCG CAT CGA GAA CCC CCC-3'; HC102, 5'-GGT TCT CGA TGC GCT TGA CCA GTA GTC TAG CAC GTG ACA GGA TCC TCC ATC AAG TAA GAT GCA GAT ACT TAA CG-biotin-3'; HC105, 5'-CTA GAC TAC TGG TCA AGC-3'; HC114, 5'-TGT ACA TAT ATC AGT GTC TG-3'; HC115, 5'-GAT GCC TCC AAG GTC GAC GAT GCA GAC ACT GAT ATA TGT ACA GAT TCG GTT GAT CAT AGC ACA ATG CCT GCT GAA CCC ACT ATC G-3'; HC116, 5'-biotin-CGA TAG TGG GTT CAG CAG GCA TTG TGC TAT GAT CAA CCG AAT CTG TAC ATA TAT CAG TGT CTG CAT CGT CGA CCT TGG AGG CAT CGG GG-3'; HC119, 5'-biotin-CGA TAG TGG GTT CAG CAG GCA TTG TGC TAT GAT CAA CCG AAT CTG TAC ATA TAT CAG TGT CTG CAT CGT CGA CCT TGG AGG CAT CTT TT-3'; JG163, 5'-GTT AAG TAT CTG CAT CTT ACT TGA CGG ATG CAA TCG TCA CGT GCT AGA CTA CTG GTC AAG CGG ATC GGG CTC GAC C-3'; JG166,

5'-CGA GCC CGA TCC GCT TGA CCA GTA GTC TAG CAC GTG ACG ATT GCA TCC GTC AAG TAA GAT GCA GAT ACT TAA CAG G-3'. Asterisks indicate phosphorothioate linkages.

Oligonucleotides used for polymerase activity test were purchased from Eurogentec and dissolved in 50 mM Tris-HCl (pH 8) and 1 mM EDTA. Concentrations were measured by UV absorbance using the absorption coefficient ε at 260 nm provided by Eurogentec. Primer strand was 5' labeled with [γ-³²P]ATP (Perkin Elmer, 3000 Ci/mM) using T4 polynucleotide kinase (New England Biolabs) for 1 hr at 37°C. The labeling reaction was stopped by heating the kinase at 75°C for 10 min. Upstream (5'-TAC GCA TTA GCC TG) and downstream (5'-P-GGC TAA TGC GTA) primers were mixed with template strand (5'-TAC GCA TTA GCC CCA GGC TAA TGC GTA), heated for 5 min up to 90°C, and slowly cooled to room temperature overnight.

## NHEJ Assay

In vitro NHEJ assays were performed as previously described (29). Briefly, NHEJ components were incubated with DNA substrates as indicated at 37°C for 1 h. Markers were generated under the same conditions. Reactions were terminated by heating at 95°C for 10 min, and samples were subsequently deproteinized using phenol-chloroform extraction. Extracted DNA was resolved using 8% denaturing PAGE and detected by autoradiography. Ligation efficiency was quantitated using Quantity One 1-D analysis software (Bio-Rad).

## Junction Sequence Analysis

Sequence analysis of ligated DNA junctions was performed as previously described (29). Briefly, DNA was visualized by exposing dried radioactive gels to an X-ray film overnight. Ligated DNA products were eluted from the gel and junction sequences were amplified from these products using
PCR primers HC105 and HC114. Amplified junction sequences were TA-cloned into pGEM-T Easy vectors (Promega) and transformed into electrocompetent DH10B cells. Transformed cells were plated on LB-agar/ampicillin/X-gal and white colonies were selected for sequencing.

## Crystallization and data collection

The dsDNA 5'-AAAAA and 5'-TTTTTGG (or 5'-TTTTTG) or gap-filling DNA (upstream primer: 5'-TGTTTG, downstream primer: 5'-CAGCG, template: 5'-CGCTGGCAAACA) were annealed in a buffer containing 50 mM Tris pH 7.8, 5 mM MgCl₂ and 2 mM EDTA. TdT-μ chimera was mixed at a final concentration of 10 mg.mL⁻¹, with ddCTP (2mM), with ddCTP (2 mM) and 2 (or 4)-fold excess of dsDNA, or with dCpCpp (2 mM) and 2-fold excess of gap-filling DNA, in a buffer containing 20 mM Tris pH 6.8, 200 mM NaCl, 100 mM ammonium sulfate and 50 mM magnesium acetate. All complexes were incubated at 4°C for 1 hour.

Crystals of TdT-μ chimera alone (apoenzyme) or mixed with ddCTP grew in 1 day at 18°C by mixing of 1 µL of concentrated protein at 10 mg.mL⁻¹ and 1 µL of mother liquor solution containing 20-24% PEG 6000, 400-800 mM lithium chloride and 100 mM MES pH 6. Crystals of TdT-μ chimera in the presence of nucleotide and dsDNA or gap-filling DNA (1 µL complex + 1 µL mother liquor) grew in 1 day at 18°C in a solution containing 19-25% PEG 4000, 100-400 mM lithium sulfate and 100 mM Tris pH 8.5. Crystals were cryo-protected using one soaking step with 25% glycerol and then flash-frozen in liquid nitrogen. X-ray data collections were collected at Soleil Synchrotron (Saint-Aubin, France) on Beamline Proxima-1 and at ESRF (Grenoble, France) on Beamlines ID23-1, ID23-2 and ID29.

## Data processing, crystallographic refinement and model validation

Diffraction datasets were processed using XDS (45) and CCP4 (46, 47). Crystals of TdT-μ chimera apoenzyme or bound to ddCTP belong to space group P2₁2₁2₁ and diffract at 2.20 Å and 1.96 Å resolution, respectively. Crystals of TdT-μ chimera in presence of 2-fold excess of dsDNA A5/T5GG and ddCTP diffract at 2.09 Å resolution and belong to space group P2₁2₁2₁. Crystals of TdT-μ chimera in the presence of a 4-fold excess of dsDNA A5/T5G and ddCTP diffract at 2.55 Å and belong to space group P2₁. Finally, crystals of TdT-μ chimera in the gap-filling mode with a continuous template strand and dCpcpp diffract to 2.35 Å and belong to space group C2 with two molecules of TdT-μ chimera per asymmetric unit. Molecular replacement was performed with the program Phaser using the 1JMS PDB file as a search model (48). Manual building by iterative cycles of model building and refinement was carried out with the software COOT (49) and BUSTER (50), using TLS parameters (51) in the last stages of refinement. The number of TLS groups was chosen by default by the program Buster. The quality of the models was assessed using MolProbity (52). Data collection and refinement statistics are reported in Table S1. Superimpositions of structures and figures were performed and generated with Chimera (53).

## Modeling Pol μ complex with the incoming dNTP and the *in trans* templating strand (dn-dsDNA)

We modeled the complete Pol μ sequence on the template of the TdT-μ chimera in a frozen backbone conformation and keeping intact the side chains of conserved residues (46%). We optimized the rotamers of the non-conserved residues globally using our Mean Field optimization algorithm implemented on our web server http://lorentz.dynstr.pasteur.fr/pdb_hydro.php (54). No major clash was observed between the modeled side-chains or with the DNA in the resulting model. The model with both the incoming dCTP (and Mg⁺⁺)
Structural model of the base selection mechanism of pol μ

and the dn-dsDNA (without the third DNA
strand) was inserted in a cubic box of
dimension such that the distance between
the protein and the edges was at least 12 Å.
The TIP3P water model was used and Na⁺
ions were added to neutralize the total
charge of the system. Force field parameters
for dCTP were obtained with CGENFF and
the CHARMM36 force field was used for
the rest of the system (55). All simulation
runs were performed using NAMD (56).
The package PSFGEN was used within
VMD (57) to build missing atoms and

create input files for NAMD. 50,000 cycles
of conjugate gradient minimization were
performed and 1000 frames were collected;
convergence occurred after about 15,000
cycles (Figure S7).

ACCESSION NUMBERS

Coordinates and structure factors have been
deposited in the PDB (http://www.rcsb.org)
with accession numbers PDB: 6GO3,
6GO4, 6GO5, 6GO6 and 6GO7 (Table S1).

ACKNOWLEDGEMENTS

We thank the staff of synchrotrons SOLEIL (Saint-Aubin, France) and ESRF (Grenoble, France) for assistance using beamlines and help during diffraction data collection. We thank L. Deriano (IP) and B. Bertocci (INSERM) for helpful discussions and B. Bertocci for the full-length constructs of TdT and Pol μ.

COMPETING INTERESTS STATEMENT

The authors declare no conflict of interest with the contents of this article.
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## FOOTNOTES

### FUNDING

This work was supported by the "Fondation ARC pour la recherche sur la cancer" through a post-doctoral fellowship to J.L. MT is supported by the PAUSE program (Collège de France, IP). Work in the lab of MRL is supported by NIH.
## Figure Legends

<caption>**Figure 1. Templated or template-independent activity of TdT, TdT-μ chimera and Pol μ in the *in cis* or *in trans* situations**</caption>

(A) Summary of the different activities of TdT-μ chimera in the presence of different DNA substrates, already known from the indicated references, illustrating its template-dependent (T-D) or template-independent (T-I) activities, both *in cis* and *in trans*. (B) Sequence alignments of Loop1 sequence in *M. musculus* TdT-wt, TdT-μ chimera and Pol μ. Residues belonging to Loop1 are represented in bold. The region SD1 is indicated in green. The special numbering choice for the insertion Q393 is highlighted in black. (C) Previously known structures of complexes of TdT (pink) or Pol μ (blue) with a primer strand and a downstream DNA duplex (D/S DNA), in a gap-filling mode, or with a full DSB-DNA junction.

<caption>**Figure 2. Ligation experiments in the presence of XRCC4:Ligase IV and Ku 70/80**</caption>

(A) Comparative functional properties of TdT wild-type, Pol μ and TdT-μ chimera using complementary or non-complementary overhangs. (B) Sequences of the products of ligation for compatible overhangs. Note that only the top strand of sequence is shown. The junctions are where the 'Additions' column is located, and when no nucleotides are present here, this means that the left and right ends are joined directly, using the compatible overhangs. (C) Sequences of the products of ligation for incompatible overhangs. Note that only the top strand of sequence is shown.

<caption>**Figure 3. Loop1 conformation in TdT wild-type, TdT-μ chimera and Pol μ without or with dNTP**</caption>

The new structures described in this paper are framed in red. No structure of the binary complex with an incoming nucleotide is available for Pol μ. Residues in Loop1 depicted in pink belong to TdT, whereas residues colored in blue belong to Pol μ. In addition, a more detailed picture of the interaction of the incoming base ddCTP with the ion pair R403-D399 (K403-D399 in TdT) of Loop1 is shown at the bottom; close distances are shown with dotted lines.

<caption>**Figure 4. Structure of TdT-μ chimera bound to a 1-nt-gapped DNA substrate with an incoming dNTP**</caption>

(A) Single-nucleotide-gapped DNA substrate: upstream and downstream primers are represented in red and blue, respectively. The continuous template strand is depicted in cyan and the incoming nucleotide is in gray. (B) Detailed view of the differences in the base moiety conformation in the ddCTP binary complex and in the gap-filling complex, accompanied by a change of rotamer in R454 and R458. (C) Overall structure of TdT-μ chimera in complex with a gap-filling DNA substrate. Loop1 (in blue) is not visible in electron density. The β2-α12 loop (junction) is indicated in grey dash lines. (D) DNA conformation in Pol μ gap-filling structure, for comparison (PDB ID: 2IHM).

<caption>**Figure 5. Ordered structure of Loop1 in TdT-μ chimera bound to the downstream DNA duplex and the incoming dNTP**</caption>

(A) Overview of the ternary complex. Loop1 is in deep blue and boxed in a red dotted frame. The DNA duplex is in black. The incoming nucleotide is in green. (B) Electron density in the 2Fo-Fc map (contoured at 1 σ in grey) in Loop1 region of TdT-μ chimera pre-ternary complex. Loop1 is represented in ball-and-stick in the context of the adjacent β3 and β4 strands, shown in grey. (C) Sequence alignments of TdT and Pol μ in Loop1 region. Loop1 of Pol μ and TdT are delimited with a light blue and pink line, respectively. The interactions between side-chain
atoms of Loop1 and the remaining part of TdT are indicated with a star (*). Strictly conserved residues are in red and residues with similar physico-chemical character are in blue.

**Figure 6. Interaction of Loop1 in the TdT-chimera with the downstream-dsDNA substrate**

(A) Loop1 conformation in TdT-μ chimera downstream-dsDNA ternary complex. The dNTP and the templating base are represented in stick.

(B) Interaction between the N-terminal part of Loop1 and TdT.

(C) Interaction between the middle part of Loop1 and TdT.

(D) Interaction between the α helix of Loop1 and TdT.

**Figure 7. Structure of TdT-chimera as a ternary complex with a downstream dsDNA and the incoming dNTP**

(A) DNA substrates: the downstream DNA duplex is colored in blue and cyan. The incoming nucleotide and Loop1 are represented in dark grey and blue, respectively. The additional DNA strand, a triplex-forming oligonucleotide (TFO) is represented in grey. (B) Space filling representation of the incoming ddNTP binding site. The ddCTP is in ball-and-stick, the downstream template strand is in cyan and the surface of the Loop1 atoms is in dark blue. (C) Overall structure of the TdT-μ chimera downstream dsDNA. The incoming ddCTP makes Watson-Crick interactions with the *in trans* template strand. Loop1 is fully visible in the electron density map and is made of two $3_{10}$ and one α helices. The exit channel for the 3' protruding end is represented in a dashed cyan line. (D) Differences in the Loop1 conformation in the binary ddCTP complex (gold) and in the down-dsDNA complex (dark blue). Loop1 conformation of TdT-WT apoenzyme is also represented as a reference (black).

**Figure 8. Structure of TdT-chimera bound to the full DNA synapsis and incoming dNTP**

(A) DNA substrates: the downstream DNA duplex is colored in blue and cyan and the upstream duplex is in red and yellow (primer strand and template strand). Incoming nucleotide and Loop1 are represented in grey and blue, respectively. The two additional DNA strands, a triplex-forming oligonucleotide (TFO), are represented in grey. (B) Overall structure of TdT-μ chimera full DNA synapsis complex. The incoming dCTP makes Watson-Crick interactions with *in trans* template strand. Loop1 is mostly invisible in the electron density map and represented in blue dash lines.

**Figure 9. Sequential model of Pol μ activity in the presence of a DSB DNA with 3' protruding ends.**

Initially, a complex composed of free Pol μ (apoenzyme) and dNTP is formed. The binding of downstream dsDNA, strengthened by the 5' phosphate binding pocket, modifies Loop1 conformation to favor Watson-Crick interactions in the nascent base-pair but also prevents the binding of upstream dsDNA, including the primer. Subsequently, Loop1 is moved away and the upstream dsDNA is recruited (DSB full synaptic complex), allowing nucleotide incorporation on the upstream primer (DSB post-catalytic complex). Finally, the enzyme and the bridged-DNA dissociate to allow for the action of Ligase IV. If the incoming dNTP does not form a Watson-Crick base pair with the downstream template DNA end, then it is possible that the ternary complex (downstream duplex + dNTP-pol μ) will disassemble. If the complex does not fall apart and the mismatched nucleotide is incorporated, then this would account for the low level of template-independent addition that is seen in Figure 2C (bottom 2 boxes).
<caption>Figure 1</caption>

A

<table><thead><tr><th rowspan="2">DNA<br>substrates</th><th colspan="2">TdT</th><th colspan="2">TdT chimera</th><th colspan="2">Pol μ</th></tr><tr><th>T-D activity</th><th>T-I activity</th><th>T-D activity</th><th>T-I activity</th><th>T-D activity</th><th>T-I activity</th></tr></thead><tbody><tr><td>In cis<br>5'</td><td>-</td><td>++<br>Romain et al., 2009</td><td>+ (Mg<sup>2+</sup>)</td><td>+ (Co<sup>2+</sup>)<br>Romain et al., 2009</td><td>++</td><td>+ (Mn<sup>2+</sup>)<br>Dominguez et al., 2000</td></tr><tr><td>In trans<br>5'</td><td>+*<br>Loc'h et al., 2016</td><td>+*<br>Loc'h et al., 2016</td><td>++<br>Loc'h et al., 2016</td><td>-</td><td>++<br>Loc'h et al., 2016</td><td>-</td></tr></tbody></table>

<figure><img src="image_2.png" /></figure>

<figure><img src="image_3.png" /></figure>

<figure><img src="image_4.png" /></figure>

<footnote>* In trans T-D vs T-DI activity in TdT is modulated by the downstream DNA concentration</footnote>
<figure><img src="image_5.png" /><figcaption>Figure 3</figcaption></figure>
<figure><img src="image_6.png" /><figcaption>Figure 4</figcaption></figure>
<figure><img src="image_7.png" /><figcaption>Figure 5</figcaption></figure>
<figure><img src="image_8.png" /><figcaption>Figure 6</figcaption></figure>
<figure><img src="image_9.png" /><figcaption>Figure 7</figcaption></figure>
<figure><img src="image_10.png" /><figcaption>Figure 8</figcaption></figure>
<figure><img src="image_11.png" /><figcaption>Figure 9</figcaption></figure>
Structural evidence for an *in trans* base selection mechanism involving Loop1 in
Polymerase mu at an NHEJ double-strand break junction

Jérôme Loc'h, Christina A. Gerodimos, Sandrine Rosario, Mustafa Tekpinar, Michael R. Lieber and Marc Delarue

J. Biol. Chem. published online May 28, 2019

Access the most updated version of this article at doi: 10.1074/jbc.RA119.008739

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