Photocross-linking of an Oriented DNA Repair Complex (1999)

Ku protein binds broken DNA ends, triggering a double-strand DNA break repair pathway. The spatial arrangement of the two Ku subunits in the initial Ku-DNA complex, when the Ku protein first approaches the broken DNA end, is not well defined. We have investigated the geometry of the complex using a novel set of photocross-linking probes that force Ku protein to be constrained in position and orientation, relative to a single free DNA end. Results suggest that this complex is roughly symmetric and that both Ku subunits make contact with an approximately equal area of the DNA. The complex has a strongly preferred orientation, with Ku70-DNA backbone contacts located proximal and Ku80-DNA backbone contacts located distal to the free end. Ku70 also contacts functional groups in the major groove proximal to the free end. Ku80 apparently does not make major groove contacts. Results are consistent with a model where the Ku70 and Ku80 subunits contact the major and minor grooves of DNA, respectively. Ku protein binds broken DNA ends, triggering a double-strand DNA break repair pathway. The spatial arrangement of the two Ku subunits in the initial Ku-DNA complex, when the Ku protein first approaches the broken DNA end, is not well defined. We have investigated the geometry of the complex using a novel set of photocross-linking probes that force Ku protein to be constrained in position and orientation, relative to a single free DNA end. Results suggest that this complex is roughly symmetric and that both Ku subunits make contact with an approximately equal area of the DNA. The complex has a strongly preferred orientation, with Ku70-DNA backbone contacts located proximal and Ku80-DNA backbone contacts located distal to the free end. Ku70 also contacts functional groups in the major groove proximal to the free end. Ku80 apparently does not make major groove contacts. Results are consistent with a model where the Ku70 and Ku80 subunits contact the major and minor grooves of DNA, respectively. DNA-dependent protein kinase catalytic subunit 70-kDa subunit of Ku 83-kDa subunit of Ku polyacrylamide gel electrophoresis nucleotide 4-morpholinepropanesulfonic acid Ku protein has a general role in the metabolism of DNA ends in eukaryotic cells, including the repair of double-strand DNA breaks (reviewed in Refs. 1Kanaar R. Hoeijmakers J.H. van Gent D.C. Trends Cell Biol. 1998; 8: 483-489Abstract Full Text Full Text PDF PubMed Scopus (454) Google Scholar and 2Critchlow S.E. Jackson S.P. Trends Biochem. Sci. 1998; 23: 394-398Abstract Full Text Full Text PDF PubMed Scopus (485) Google Scholar). Ku binds tightly to broken ends and initiates a repair pathway. Biochemical studies in higher eukaryotes, as well as genetic analysis in yeast, have helped to identify many participants in this pathway. In mammalian cells, these include a protein kinase (DNA-PKcs),1 a ligase (DNA ligase IV), a nuclease (hMRE11), and various protein cofactors (XRCC4, hRAD50, p95/NBS1) (reviewed in Ref. 1Kanaar R. Hoeijmakers J.H. van Gent D.C. Trends Cell Biol. 1998; 8: 483-489Abstract Full Text Full Text PDF PubMed Scopus (454) Google Scholar). One of the functions of Ku protein is to recruit the 470-kDa DNA-dependent protein kinase catalytic subunit (DNA-PKcs) to DNA (3Dvir A. Peterson S.R. Knuth M.W. Lu H. Dynan W.S. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 11920-11924Crossref PubMed Scopus (337) Google Scholar, 4Gottlieb T.M. Jackson S.P. Cell. 1993; 72: 131-142Abstract Full Text PDF PubMed Scopus (1026) Google Scholar). However, in a number of instances, Ku has been shown to have independent functions in repair that do not require DNA-PKcs. These include the alignment of DNA ends in noncovalent complexes visible in the atomic force microscope and the stimulation of eukaryotic DNA ligases (5Pang D. Yoo S. Dynan W.S. Jung M. Dritschilo A. Cancer Res. 1997; 57: 1412-1415PubMed Google Scholar, 6Ramsden D.A. Gellert M. EMBO J. 1998; 17: 609-614Crossref PubMed Scopus (246) Google Scholar). Studies with mutant cell lines further support the idea that there are functions of Ku that are independent of DNA-PKcs. For example, cells lacking Ku are hypersensitive to the action of the double-strand DNA break-inducing topoisomerase II inhibitor, etoposide, whereas cells lacking DNA-PKcs are not (7Jin S. Inoue S. Weaver D.T. Carcinogenesis. 1998; 19: 965-971Crossref PubMed Scopus (54) Google Scholar). Cells lacking Ku are unable to perform either V(D)J coding or signal joint formation (8Smider V. Rathmell W.K. Lieber M.R. Chu G. Science. 1994; 266: 288-291Crossref PubMed Scopus (320) Google Scholar, 9Taccioli G.E. Gottlieb T.M. Blunt T. Priestley A. Demengeot J. Mizuta R. Lehmann A.R. Alt F.W. Jackson S.P. Jeggo P.A. Science. 1994; 265: 1442-1445Crossref PubMed Scopus (591) Google Scholar), whereas most cells lacking DNA-PKcs remain competent to perform signal joint formation (10Lieber M.R. Hesse J.E. Lewis S. Bosma G.C. Rosenberg N. Mizuuchi K. Bosma M.J. Gellert M. Cell. 1988; 55: 7-16Abstract Full Text PDF PubMed Scopus (368) Google Scholar, 11Gao Y. Chaudhuri J. Zhu C. Davidson L. Weaver D.T. Alt F.W. Immunity. 1998; 9: 367-376Abstract Full Text Full Text PDF PubMed Scopus (362) Google Scholar, 12Kurimasa A. Ouyang H. Dong L.J. Wang S. Li X. Cordon-Cardo C. Chen D.J. Li G.C. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1403-1408Crossref PubMed Scopus (159) Google Scholar). Finally, Ku homologues have been identified in a variety of fungal and metazoan organisms, including yeast, roundworms, insects, and vertebrates. By contrast, DNA-PK activity has so far been detected only in one branch of the higher eukaryotic evolutionary tree (the Coelomata), and, among these, the DNA-PKcs gene or polypeptide has been identified unequivocally only in the taxon that includes echinoderms and vertebrates (the Deuterostoma) (reviewed in Ref. 13Dynan W.S. Yoo S. Nucleic Acids Res. 1998; 26: 1551-1559Crossref PubMed Scopus (297) Google Scholar). It thus appears that DNA-PKcs appeared later in evolution than Ku itself. Because of the central and evolutionarily ancient role of Ku protein in double-strand DNA break repair, it is important to understand the mechanism by which it recognizes DNA ends. Human Ku is a heterodimer of 70- and 83-kDa subunits, neither of which contains known DNA binding motifs. In vitro binding studies show that the interaction of Ku with DNA is determined primarily by features of the nucleic acid structure (13Dynan W.S. Yoo S. Nucleic Acids Res. 1998; 26: 1551-1559Crossref PubMed Scopus (297) Google Scholar). Ku binds blunt, overhanging, and hairpin ends (14Mimori T. Hardin J.A. Steitz J.A. J. Biol. Chem. 1986; 261: 2274-2278Abstract Full Text PDF PubMed Google Scholar, 15Falzon M. Fewell J.W. Kuff E.L. J. Biol. Chem. 1993; 268: 10546-10552Abstract Full Text PDF PubMed Google Scholar, 16Paillard S. Strauss F. Nucleic Acids Res. 1991; 19: 5619-5624Crossref PubMed Scopus (188) Google Scholar). End-bound Ku can translocate inward, forming a sliding clamp (17Zhang W.W. Yaneva M. Biochem. Biophys. Res. Commun. 1992; 186: 574-579Crossref PubMed Scopus (76) Google Scholar). In addition to DNA ends, Ku is capable of binding artificial DNA structures containing single- to double-strand transitions (15Falzon M. Fewell J.W. Kuff E.L. J. Biol. Chem. 1993; 268: 10546-10552Abstract Full Text PDF PubMed Google Scholar, 18Blier P.R. Griffith A.J. Craft J. Hardin J.A. J. Biol. Chem. 1993; 268: 7594-7601Abstract Full Text PDF PubMed Google Scholar). There are also certain sequences that appear to function as entry sites for Ku in double-stranded DNA (19Torrance H. Giffin W. Rodda D.J. Pope L. Hache R.J. J. Biol. Chem. 1998; 273: 20810-20819Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). Previous studies have shown different requirements for the Ku70 and Ku80 subunits in DNA end binding, depending on the assay used. Ku70 binds DNA in the absence of Ku80 in immunoprecipitation and Southwestern blot analysis (20Wang J. Satoh M. Chou C.H. Reeves W.H. FEBS Lett. 1994; 351: 219-224Crossref PubMed Scopus (26) Google Scholar, 21Wang J. Dong X. Myung K. Hendrickson E.A. Reeves W.H. J. Biol. Chem. 1998; 273: 842-848Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 22Wang J. Dong X. Reeves W.H. J. Biol. Chem. 1998; 273: 31068-31074Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). On the basis of these results, it has been suggested that the major part of the Ku DNA binding domain may reside in the Ku70 subunit. However, DNA binding activity requires sequences in the C-terminal half of both Ku subunits in an electrophoretic mobility shift assay (23Wu X. Lieber M.R. Mol. Cell. Biol. 1996; 16: 5186-5193Crossref PubMed Scopus (110) Google Scholar). Furthermore, sequence analysis reveals that Ku70 and Ku80 share weak homology, most evident in lower eukaryotes, over a large C-terminal region encompassing the DNA binding domains defined in electrophoretic mobility shift assay (13Dynan W.S. Yoo S. Nucleic Acids Res. 1998; 26: 1551-1559Crossref PubMed Scopus (297) Google Scholar). This suggests that the subunits derive from duplication of a common ancestral gene and are likely to share a similar tertiary folding pattern. Consistent with this, recent studies have shown that the sequences required for dimerization map to precisely homologous positions in each subunit of Ku (21Wang J. Dong X. Myung K. Hendrickson E.A. Reeves W.H. J. Biol. Chem. 1998; 273: 842-848Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 22Wang J. Dong X. Reeves W.H. J. Biol. Chem. 1998; 273: 31068-31074Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 24Osipovich O. Durum S.K. Muegge K. J. Biol. Chem. 1997; 272: 27259-27265Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). The existence of sequence homology in Ku70 and Ku80 suggests, but does not prove, that both subunits may have equivalent roles in contacting DNA. We have performed site-specific cross-linking experiments to distinguish between the different models for the role of the two Ku subunits. These experiments define the spatial arrangement of the Ku subunits in the Ku-DNA repair complexes assembled on defined oligonucleotides. Some cross-linking studies with Ku have been performed before (4Gottlieb T.M. Jackson S.P. Cell. 1993; 72: 131-142Abstract Full Text PDF PubMed Scopus (1026) Google Scholar, 17Zhang W.W. Yaneva M. Biochem. Biophys. Res. Commun. 1992; 186: 574-579Crossref PubMed Scopus (76) Google Scholar, 19Torrance H. Giffin W. Rodda D.J. Pope L. Hache R.J. J. Biol. Chem. 1998; 273: 20810-20819Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar, 25Knuth M.W. Gunderson S.I. Thompson N.E. Strasheim L.A. Burgess R.R. J. Biol. Chem. 1990; 265: 17911-17920Abstract Full Text PDF PubMed Google Scholar, 26Yaneva M. Kowalewski T. Lieber M.R. EMBO J. 1997; 16: 5098-5112Crossref PubMed Scopus (267) Google Scholar). In particular, a report by Zhang and Yaneva (17Zhang W.W. Yaneva M. Biochem. Biophys. Res. Commun. 1992; 186: 574-579Crossref PubMed Scopus (76) Google Scholar) indicated the relative positions of Ku70 and Ku80 subunit at the DNA end, based on the pattern of protection of probes that were radiolabeled by different methods. However, this early study relied on nonspecific UV cross-linking chemistry and did not involve the introduction of defined cross-linking groups at specific positions in the DNA. In the present work, we have synthesized a novel set of probes that force Ku to be trapped in a unique position and orientation relative to a single free DNA end. Incorporation of aryl azide groups at a number of different backbone positions allows unambiguous determination of the proximity of Ku70 and Ku80 to different points on the DNA surface. The results of these studies show that Ku protein binds DNA as a roughly symmetric, oriented heterodimer, with each subunit making approximately equal numbers of backbone contacts with DNA. Ku70 makes contacts functional groups in the major groove of DNA, while Ku80 apparently does not. To express non-histidine-tagged Ku70, a DNA fragment encoding histidine-tagged Ku70 was excised from pBC-70tH6 (27Ono M. Tucker P.W. Capra J.D. Nucleic Acids Res. 1994; 22: 3918-3924Crossref PubMed Scopus (72) Google Scholar) with BamHI and subcloned into pGEM3zf(+) (Promega, Madison, WI). The open reading frame, without the histidine was by using as and The fragment was with BamHI and subcloned into non-histidine-tagged Ku was by of cells with this and DNA Ku was from cell as that the and were A. Dynan W.S. J. Biol. Chem. 1993; 268: Full Text PDF PubMed Google Scholar). To Ku cells were with (27Ono M. Tucker P.W. Capra J.D. Nucleic Acids Res. 1994; 22: 3918-3924Crossref PubMed Scopus (72) Google Scholar) and either or with a of of cells were and in of containing The was with for on to and for at To Ku without a histidine were from of with and was to the and the was at for The was by at in of and at for The was to a and in with containing Ku were to and to a single Ku was with a of in were to to a in and with of and in Ku by this is functional in kinase and mobility shift not Ku with a histidine was as J. A. Yoo S. Dynan W.S. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). To probes without cross-linking a and a with a were synthesized with the sequence shown in The were and radiolabeled using and To a was synthesized that a and the This was to a The DNA was and radiolabeled using and were by DNA and at for in and Ku was and to for were by polyacrylamide gel electrophoresis in a containing and To DNA probes probes with a single were synthesized and to DNA containing a at the end. were gel and in a containing and were radiolabeled at the end of the using and To DNA probes probes were synthesized that the that a single and that a at the end. These were to DNA, and radiolabeled by with and were for in the at in a containing DNA, and were using complexes were in that were a cell The were with for were by addition of and for at were by and detected by containing of or were synthesized The were to DNA containing a were and as was in for using a a double-strand DNA break in each fragment has one broken end. These ends an entry for Ku and repair The repair complex has an orientation, with proximal and distal to the free DNA end. We to probes that the in repair complexes can at either of the two ends of a when contacts. double-stranded DNA probes with a a at the end of one In the of one DNA end is the end to the of repair complex it is likely that Ku protein makes initial contacts with the DNA ends, it is known to translocate the DNA in an In to the Ku at a unique position to the initial complex, it was to identify the required for Ku To this end, a of in was for formation of a complex with and was the to a complex, shown in A. This complex was from and Ku-DNA complexes To the of in the DNA ends, a with at both ends was also In the absence of this DNA a complex with as In the of Ku did not to this that did both ends. these suggest that the can be to map the spatial arrangement of Ku70 and Ku80 in an oriented complex when the Ku protein recognizes the broken DNA end. set of probes was each with a single aryl azide at a different was to backbone position on either of the DNA. each aryl azide is capable of with a Dynan W.S. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar), this of the DNA surface. were to to and with were by a cross-linking using a the of the which allows contact with both Ku subunits. well radiolabeled were and to Ku70 and Ku80 Ku70 and Ku80 are similar in it was important to the cross-linking to the two subunits, than cross-linking to the subunit at different sites to of To distinguish these the one containing a Ku70 subunit with a histidine and This a mobility shift of the lower relative to Ku and Ku This shift is with that with of these and the lower as a Ku70 The has the mobility in and is a Ku80 For cross-linking Ku protein was used. We the set of the we performed a electrophoretic mobility shift which that of the aryl azide with Ku-DNA binding the not complexes were by In the absence of and we to a of complexes assembled in both on the DNA. Consistent with this, these the of the probes to Ku70 and Ku80 in approximately equal probes either a or for Ku70 over Ku80 was present to one end of the DNA and there was a in the pattern of Ku-DNA these there were probes that and in to and The of these probes are by in the in C. are the distal half of the DNA, relative to the free end, and define a region of Ku80 There were two and that did not to either subunit of Ku in the of These define a region of the of the on the There were probes that not to Ku70 in the of and The of these probes are by C. the proximal half of the DNA, relative to the free end, and define a region that is primarily by Ku70 and, to a by these results show that Ku in a defined orientation relative to the free DNA end and that contacts with Ku70 and Ku80 are roughly symmetric an to the DNA of the cross-linking results further and In these relative cross-linking to different positions on the is indicated by the and cross-linking to equivalent positions on the is indicated by the cross-linking of There is a of sites proximal to the free end. the sites of cross-linking are not each on the and but are by in the on each on the and on the In DNA, the that most each on of the major groove are by in the on each a pattern of contacts is strongly of a protein binding that the major groove M.R. C. Steitz Proc. Natl. Acad. Sci. U. S. A. 1990; PubMed Scopus Google Scholar). There are two of Ku80 cross-linking in One in the distal half of the fragment relative to the free end, of probes that only to Ku80 this distal the sites of cross-linking on the two are by in the Because the that most each the minor groove of DNA are by to the on each a pattern of contacts is strongly of a binding that the minor of Ku80 cross-linking is located in the proximal half of the fragment is than in the distal and of these probes also to Ku70 strongly than to There are of the Ku80 cross-linking with these One is that the are to with Ku80 in the distal is that Ku80 sequences are with Ku70 in the Ku-DNA contacts were further investigated using DNA with acid that are in contact with the position of or in the major groove of DNA Biochem. Mol. Biol. 1997; PubMed Scopus Google Scholar). Because Ku is a we that contacts with depending on the sequence that is present in the was to probes where were This the that a of Ku that the major groove make at one contact with a was performed in the and absence of and results are shown in A. In the absence of Ku70 to probes with the ends of the DNA and but not to a with in the In the of to the complexes Ku70 only to a at positions the free end and not to the These results are consistent with the aryl azide and that Ku70 makes contacts with the major groove at one or sites the proximal half of the relative to the free end. Ku80 did not to of the probes that were This is consistent with the that Ku80 binds a that the minor than the major the interaction of Ku protein with nucleic has been the of Ku-DNA contacts remain One of the is Ku can to either end of a DNA there is a in the orientation of the Ku-DNA Ku is also known to translocate the DNA, the pattern of contacts. In the present we have a cross-linking that has been to study DNA This is to the geometry of In this aryl azide cross-linking groups are at specific DNA We and synthesized a set of novel aryl probes in which one end of the DNA is the Ku protein to to the end of DNA, in a specific we have the that formation of a Ku-DNA complex, the Ku from sliding the DNA and also that a of the contacts are likely to be This has to a map of the Ku-DNA the proximity of each subunit to different points on the DNA surface. of the DNA in this as DNA, is shown in The sites of of the aryl azide groups that to Ku70 and Ku80 are shown in and respectively. The position of that to Ku70 are shown To the of the Ku-DNA the sites of cross-linking are by the and the numbers are indicated in It appears that the sites of Ku70 contact are in a in the major groove defined by probes and The also this the aryl azide probes have a a Ku70 the major groove be with probes at including and both the of the backbone contacts and the with are consistent with a major groove binding model for The sites of Ku80 contact appear to to a region of the minor groove defined by probes and the shown in this region the DNA to the Ku80 in this region be with consistent with the pattern of minor cross-linking probes are not both the of the backbone contacts and the absence of are consistent with minor groove for It is that Ku-DNA contacts are located primarily on one of the This is from of where the DNA model has been relative to the in A. The Ku70 binding to the and the Ku80 binding to and In this there are contacts on the of the and show cross-linking to probes and is also The arrangement of Ku protein on one of the allows repair to the DNA including the or the DNA ligase The that each Ku subunit contacts DNA to a similar in the Ku-DNA complex in to an that the interaction of Ku with DNA primarily on the Ku70 subunit (20Wang J. Satoh M. Chou C.H. Reeves W.H. FEBS Lett. 1994; 351: 219-224Crossref PubMed Scopus (26) Google Scholar, 21Wang J. Dong X. Myung K. Hendrickson E.A. Reeves W.H. J. Biol. Chem. 1998; 273: 842-848Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 22Wang J. Dong X. Reeves W.H. J. Biol. Chem. 1998; 273: 31068-31074Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). was based on that Ku70, but not binds DNA in immunoprecipitation and Southwestern blot It that both subunits to binding in the Ku The of sequence homology between the two subunits suggest that both may share a common folding pattern. results that at the of the DNA contacts are different for the two subunits. Ku70 binds in the major whereas Ku80 apparently binds in the minor The between major and minor grooves at the of the complex and is in for Ku to remain primarily on one of a recent study a structure of Ku-DNA complexes using a J. Wang J. Chen D.J. Peterson S.R. J. 1999; PubMed Scopus Google Scholar). These and suggested that these a that into the Ku This model is at with cross-linking which strongly suggests a region of on one of the DNA. the in the make one is that the complex between Ku and the an initial interaction with the DNA end and that of Ku into contact with DNA when a sequence is contacts do not appear to the Ku-DNA binding as both in and in a study (15Falzon M. Fewell J.W. Kuff E.L. J. Biol. Chem. 1993; 268: 10546-10552Abstract Full Text PDF PubMed Google Scholar). It is that the Ku-DNA contacts may a functional role in the of higher complexes containing of Ku repair studies have identified a number of that in a repair S.E. Jackson S.P. Trends Biochem. Sci. 1998; 23: 394-398Abstract Full Text Full Text PDF PubMed Scopus (485) Google Scholar), and homologues of most of these are The using site-specific DNA probes with a single free end, is to the study of higher repair using to the of repair the geometry of the double-strand break repair complex to be further We for of Ku and for of cells, of for the DNA and for a reading of the