Accurate in Vitro End Joining of a DNA Double Strand Break with Partially Cohesive 3′-Overhangs and 3′-Phosphoglycolate Termini

To examine determinants of fidelity in DNA end joining, a substrate containing a model of a staggered free radical-mediated double-strand break, with cohesive phosphoglycolate-terminated 3′-overhangs and a one-base gap in each strand, was constructed. In extracts of Xenopus eggs, human lymphoblastoid cells, hamster CHO-K1 cells, and a Chinese hamster ovary (CHO) derivative lacking the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs), the predominant end joining product was that corresponding to accurate restoration of the original sequence. In extracts of the Ku-deficient CHO derivative xrs6, a shorter product, consistent with 3′ → 5′ resection before ligation, was formed. Similar results were seen for a substrate with 5′-overhangs and recessed 3′-phosphoglycolate ends. Supplementation of the xrs6 extracts with purified Ku restored accurate end joining. In Xenopus and human extracts, but not in hamster extracts, gap filling and ligation were blocked by wortmannin, consistent with a requirement for DNA-PKcs activity. The results suggest a Ku-dependent pathway, regulated by DNA-PKcs, that can accurately restore the original DNA sequence at sites of free radical-mediated double-strand breaks, by protecting DNA termini from degradation and maintaining the alignment of short partial complementarities during gap filling and ligation. To examine determinants of fidelity in DNA end joining, a substrate containing a model of a staggered free radical-mediated double-strand break, with cohesive phosphoglycolate-terminated 3′-overhangs and a one-base gap in each strand, was constructed. In extracts of Xenopus eggs, human lymphoblastoid cells, hamster CHO-K1 cells, and a Chinese hamster ovary (CHO) derivative lacking the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs), the predominant end joining product was that corresponding to accurate restoration of the original sequence. In extracts of the Ku-deficient CHO derivative xrs6, a shorter product, consistent with 3′ → 5′ resection before ligation, was formed. Similar results were seen for a substrate with 5′-overhangs and recessed 3′-phosphoglycolate ends. Supplementation of the xrs6 extracts with purified Ku restored accurate end joining. In Xenopus and human extracts, but not in hamster extracts, gap filling and ligation were blocked by wortmannin, consistent with a requirement for DNA-PKcs activity. The results suggest a Ku-dependent pathway, regulated by DNA-PKcs, that can accurately restore the original DNA sequence at sites of free radical-mediated double-strand breaks, by protecting DNA termini from degradation and maintaining the alignment of short partial complementarities during gap filling and ligation. DNA-dependent protein kinase catalytic subunit of DNA-PK double strand break phosphoglycolate Chinese hamster ovary dithiothreitol base pair(s) Cells deficient in any of the three components of DNA-dependent protein kinase DNA-PK1 (i.e. the catalytic subunit DNA-PKcs or either subunit of the DNA end binding heterodimer Ku) are partially deficient in the rejoining of double strand breaks (DSBs) (1Chu G. J. Biol. Chem. 1997; 272: 24097-24100Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar, 2Lieber M.R. Grawunder U. Wu X. Yaneva M. Curr. Opin. Genet. Dev. 1997; 7: 99-104Crossref PubMed Scopus (127) Google Scholar, 3Lees-Miller S.P. Biochem. Cell Biol. 1996; 74: 503-512Crossref PubMed Scopus (109) Google Scholar, 4Jeggo P.A. Mutat. Res. 1997; 384: 1-14Crossref PubMed Scopus (161) Google Scholar). The precise roles of the individual subunits in the repair process are not known, but possible functions have been suggested based on their known biochemical properties. Ku has been proposed to align DNA ends and thus promote ligation (5Yaneva M. Kowalewski T. Lieber M.R. EMBO J. 1997; 16: 5098-5112Crossref PubMed Scopus (271) Google Scholar, 6Cary R.B. Peterson S.R. Wang J. Bear D.G. Bradbury E.M. Chen D.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4267-4272Crossref PubMed Scopus (228) Google Scholar, 7Pang D. Yoo S. Dynan W.S. Jung M. Dritschilo A. Cancer Res. 1997; 57: 1412-1415PubMed Google Scholar), to protect the ends from degradation (8Feldmann E. Schmiemann V. Goedecke W. Reichenberger S. Pfeiffer P. Nucleic Acids Res. 2000; 28: 2585-2596Crossref PubMed Scopus (191) Google Scholar), and/or to unwind duplex DNA ends and thus expose microhomologies that could be used for splicing the ends together (1Chu G. J. Biol. Chem. 1997; 272: 24097-24100Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar, 9Featherstone C. Jackson S.P. Mutat. Res. 1999; 434: 3-15Crossref PubMed Scopus (240) Google Scholar). DNA-PKcs has been proposed to regulate accessibility of DNA ends to processing (10Gu X.-Y. Bennett R.A.O. Povirk L.F. J. Biol. Chem. 1996; 271: 19660-19663Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar), perhaps by promoting its own dissociation following autophosphorylation (11Chan D.W. Lees-Miller S.P. J. Biol. Chem. 1996; 271: 8936-8941Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar) and/or by allowing translocation of Ku from the ends into the interior of the DNA (10Gu X.-Y. Bennett R.A.O. Povirk L.F. J. Biol. Chem. 1996; 271: 19660-19663Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 12Calsou P. Frit P. Humbert O. Muller C. Chen D.J. Salles B. J. Biol. Chem. 1999; 274: 7848-7856Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar,13Smith G.C. Divecha N. Lakin N.D. Jackson S.P. Biochem. Soc. Symp. 1999; 64: 91-104PubMed Google Scholar). Ionizing radiation is a major environmental source of DSBs (14Ward J.F. Prog. Nucleic Acids Res. Mol. Biol. 1988; 35: 95-125Crossref PubMed Scopus (1233) Google Scholar, 15Hutchinson F. Prog. Nucleic Acids Res. Mol. Biol. 1985; 32: 115-154Crossref PubMed Scopus (463) Google Scholar, 16Obe G. Johannes C. Schulte-Frohlinde D. Mutagenesis. 1992; 7: 3-12Crossref PubMed Scopus (149) Google Scholar). Radiation-induced DSBs are formed by fragmentation of deoxyribose, typically leaving in each strand a one-base gap with 5′-phosphate and either 3′-phosphate or 3′-phosphoglycolate (PG) termini (14Ward J.F. Prog. Nucleic Acids Res. Mol. Biol. 1988; 35: 95-125Crossref PubMed Scopus (1233) Google Scholar, 15Hutchinson F. Prog. Nucleic Acids Res. Mol. Biol. 1985; 32: 115-154Crossref PubMed Scopus (463) Google Scholar, 17Henner W.D. Rodriguez L.O. Hecht S.M. Haseltine W.A. J. Biol. Chem. 1983; 258: 711-713Abstract Full Text PDF PubMed Google Scholar). Such DSBs present to repair systems a more complex substrate than simple restriction enzyme cuts, potentially increasing the possibility of errors during rejoining. In this report, we describe how synthetic substrates containing mimics of radiation-induced breaks, with defined geometry and chemical structure (see Figs. 1 and 2), are processed in several in vitro end joining systems. The results suggest a Ku-dependent repair process that can accurately restore the original DNA sequence at sites of complex the of short complementarities at the ends of the joining substrates and repair the substrate mimics the that from free radical-mediated fragmentation on a 3′ repair to ligation. the substrate was a to the of ligation in of the In any product, but could a product by of the of the and of the a and from of and from the to the substrate the but is to a free radical-mediated three substrates by of and/or be to with the alignment of in the repair CHO and derivative were from the of Cell for A. B. F. M. Jackson S.P. Nucleic Acids Res. PubMed Scopus (56) Google Scholar), were from M. of Xenopus Pfeiffer P. M. P. A. PubMed Scopus Google Scholar), Chinese hamster P. Reichenberger S. B. M. E. Pfeiffer P. Biol. Chem. 1996; PubMed Scopus Google Scholar), and human lymphoblastoid P. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar) were to that for Xenopus extracts, the was 1 1 1 1 1 1 the a M. Jackson S.P. J. Cell Sci. 1996; Google Scholar). substrate containing a DNA double strand break with cohesive 5′-overhangs and recessed termini was by ligation of into 5′-overhangs formed by of with the of (10Gu X.-Y. Bennett R.A.O. Povirk L.F. J. Biol. Chem. 1996; 271: 19660-19663Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, R.A.O. X.-Y. Povirk L.F. J. Biol. 1996; PubMed Scopus Google Scholar). but a was by ligation of and the a into the The structure of the was by could be to or of the or to the with S. Povirk L.F. Biochem. 2000; Scopus Google Scholar). R.A.O. X.-Y. Povirk L.F. J. Biol. 1996; PubMed Scopus Google Scholar), the was of any termini by with DNA in the of in the of the substrate with a to in of a substrate with a this was from the and in to that repair were from that 3′-overhangs into were with a into a been at end Ku protein was purified D.W. Lees-Miller S.P. Biochem. Cell Biol. 1996; 74: PubMed Scopus Google Scholar). with extracts of CHO-K1 and CHO of and with a protein of of substrate in a of were at for (8Feldmann E. Schmiemann V. Goedecke W. Reichenberger S. Pfeiffer P. Nucleic Acids Res. 2000; 28: 2585-2596Crossref PubMed Scopus (191) Google Scholar). of and of DNA were at for P. W. Nucleic Acids Res. 1988; 16: PubMed Scopus Google Scholar). with extracts of human 1 substrate and at a protein of and were at for P. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). in was to to a of and/or the end joining were with and and were and and to on M. Povirk L.F. PubMed Scopus Google Scholar). were and on for at were with were the of of DNA were on at of the and end joining were for of of the were into by the were in and was in the corresponding to the by the The was base from the Radiation-induced DNA DSBs are formed from a and in J.F. Res. PubMed Scopus Google Scholar). To examine the repair of by end joining, a model was end of the break a with the sequence this substrate mimics the break that from free radical-mediated at each in the sequence repair of a break of the in each of of the gap in each strand the from the end of the break a and ligation. In that processing of the break could be was from of the 3′ substrate was in CHO-K1 extracts and with restriction on each end to short were on in the CHO-K1 extracts were to breaks, and the predominant product was a corresponding to accurate rejoining by gap and ligation, of repair in individual from the repair the sequence of this repair its repair In a product could be formed from the joining of each of the To this were with a substrate into the of the substrate The end of this have a formed in the (see R.A.O. X.-Y. Povirk L.F. J. Biol. 1996; PubMed Scopus Google Scholar). this not any repair product, but a of with of and The product to of the and of the by ligation of the ends. The and to of and of the of the was by of The and to of and of the on of the break, by a or 3′ resection of the end with a the to ligation of the into the or of the be from the of accurate for the is than is is and The of repair by the is repair from into the been but not be in the of repair formed in hamster extracts from the and sequence is in of the strand in The the from ends of the break, can be from the sequence. the of the strand of the that be of in the of the the a that have been used for at the of the repair was → shorter than the accurate product, the break in the repair was → shorter than the accurate repair repair was → shorter than the accurate repair repair were → → and → shorter than the accurate repair The sequence is in of the strand in The the from ends of the break, can be from the sequence. the of the strand of the that be in the of the the a that have been used for at the of the The repair was → shorter than the accurate product, the break in the The repair was → shorter than the accurate repair The repair was → shorter than the accurate repair The repair were → → and → shorter than the accurate repair in a a that the product was the of gap the were with of was for of the product, the that was formed by gap filling the in each The of on the product end joining The of any to 3′ resection in extracts from of the of with of a consistent with the of in the one-base gap been in with the ligation the of with a partial of was to accurate end joining with restoration of the original of to repair corresponding to of Ku is a protein that has been in repair in (1Chu G. J. Biol. Chem. 1997; 272: 24097-24100Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar, 2Lieber M.R. Grawunder U. Wu X. Yaneva M. Curr. Opin. Genet. Dev. 1997; 7: 99-104Crossref PubMed Scopus (127) Google Scholar, 3Lees-Miller S.P. Biochem. Cell Biol. 1996; 74: 503-512Crossref PubMed Scopus (109) Google Scholar, 4Jeggo P.A. Mutat. Res. 1997; 384: 1-14Crossref PubMed Scopus (161) Google Scholar) and has been to promote DNA (5Yaneva M. Kowalewski T. Lieber M.R. EMBO J. 1997; 16: 5098-5112Crossref PubMed Scopus (271) Google Scholar, 7Pang D. Yoo S. Dynan W.S. Jung M. Dritschilo A. Cancer Res. 1997; 57: 1412-1415PubMed Google Scholar, M. EMBO J. PubMed Scopus Google Scholar) and to rejoining by DNA in vitro M. EMBO J. PubMed Scopus Google Scholar, J. Mol. Biol. 2000; PubMed Scopus Google Scholar). To the possible of Ku on the and of end joining, were extracts of the CHO derivative xrs6 P.A. Mutat. Res. 1983; PubMed Scopus Google Scholar, J. Biol. Chem. Full Text PDF PubMed Google Scholar). in and was of the accurately repair product in xrs6 the predominant product was the product, consistent with 3′ resection in each strand and of the (see In to be more 3′ processing in the xrs6 than in the CHO-K1 xrs6 extracts, ends and more than of the been to resection at several into the duplex of the in extracts, of several repair with at the break the of the product with splicing at the the of any accurate repair To the CHO-K1 and were to or of Ku or to the extracts were from derivative that been with a hamster S. J. Biol. PubMed Scopus Google Scholar). xrs6 extracts, extracts were to the accurately repair product and the of purified human Ku restored accurate repair in the xrs6 extracts, promoting of the product of the product the of end joining was restored to that of CHO-K1 extracts, the was with joining in the joining in The for this is not known, but could in Ku the human and hamster or of Ku on in the the results suggest a and requirement for Ku in the accurate end joining of free radical-mediated Similar were with a substrate ends and cohesive this substrate free radical-mediated not have a gap in each strand the ends were the of Ku 3′ → 5′ resection and accurate end joining, the requirement was not CHO-K1 and extracts, and of the at in the product, consistent with of the The xrs6 the product consistent with a 3′ resection by of the (see 2), but product The of purified human Ku to extracts restored accurate end joining the of end joining was that seen with the CHO-K1 of the extracts of the and joining that was was more end processing at of in xrs6 than in CHO-K1 either Ku or Ku restored accurate end joining of the substrate to To end joining DNA-PKcs, of the DNA-PKcs were the and the of end joining. at a to DNA-PK (10Gu X.-Y. Bennett R.A.O. Povirk L.F. J. Biol. Chem. 1996; 271: 19660-19663Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, M. Povirk L.F. PubMed Scopus Google Scholar, D. Divecha N. A. S.P. Jackson S.P. Full Text PDF PubMed Scopus Google Scholar), on of the and end joining and a than in the of the product To DNA-PKcs of its kinase were with extracts of the CHO derivative DNA-PKcs protein A. B. F. M. Jackson S.P. Nucleic Acids Res. PubMed Scopus (56) Google Scholar). xrs6 extracts, extracts of the end joining product that DNA-PKcs is not for accurate end joining in this In extracts of Xenopus eggs, end joining of simple restriction and can be blocked by DNA-PK and (10Gu X.-Y. Bennett R.A.O. Povirk L.F. J. Biol. Chem. 1996; 271: 19660-19663Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, M. Povirk L.F. PubMed Scopus Google end joining is in at of human extracts P. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). or on end joining of substrates in CHO or xrs6 In to the for and examine the possible of DNA-PK in joining of the were in Xenopus extracts and in extracts of human lymphoblastoid In the Xenopus extracts, the product was to that seen in CHO-K1 extracts The product was but was of the and several shorter in CHO-K1 extracts, the the and shorter that the product was formed by of the extracts, Xenopus extracts of to partial or of the (see In Xenopus extracts, of was blocked by in a to DNA-PK M. Povirk L.F. PubMed Scopus Google Scholar). joining was to in Xenopus extracts than in the extracts to the the was In the of a in the for a and could be corresponding to of the gap in the strand of the to ligation. a joining product could be formed. To were in to the product to the ligation The of either or of the end joining product and the of the results suggest that the was the but and that was blocked by wortmannin, a of of the kinase of DNA-PK M. Povirk L.F. PubMed Scopus Google Scholar), can regulate accessibility of DNA ends to processing P. Frit P. Humbert O. Muller C. Chen D.J. Salles B. J. Biol. Chem. 1999; 274: 7848-7856Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). In to a recessed (10Gu X.-Y. Bennett R.A.O. Povirk L.F. J. Biol. Chem. 1996; 271: 19660-19663Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar), was of from the the of In human extracts, a of end joining was seen The substrate the accurate end joining product, with of the and but of the The substrate the accurately product and of the product In human extracts more of the and end joining than hamster and Xenopus extracts, that was or for on P. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar), the human extracts with in extracts, of end joining was blocked by wortmannin, of for of the accurate and end joining and the but in end joining in human extracts was by the by was The of to DNA ends based on short partial complementarities in strand was in the repair formed with restriction ends were into Mol. Biol. PubMed Scopus Google Scholar). In of repair partial complementarities in 3′-overhangs been used for the that alignment strand in the ends. a the repair complex have the ends in and base of a or in the at the allowing a to in the to a vitro Xenopus extracts S. A. Pfeiffer P. W. Full Text PDF PubMed Scopus Google Scholar, P. S. J. W. Mol. Biol. PubMed Scopus Google Scholar) that processing was and thus was proposed but not alignment has been to the microhomologies typically at sequence of for D. Scholar, M. D. M. for D. Scholar), the be for the accurate repair of DSBs by radiation and free Radiation-induced breaks are the of free radical-mediated fragmentation J.F. Res. PubMed Scopus Google Scholar), and are to be staggered and to have cohesive of to several with blocked 3′ termini (14Ward J.F. Prog. Nucleic Acids Res. Mol. Biol. 1988; 35: 95-125Crossref PubMed Scopus (1233) Google Scholar, 15Hutchinson F. Prog. Nucleic Acids Res. Mol. Biol. 1985; 32: 115-154Crossref PubMed Scopus (463) Google Scholar, 17Henner W.D. Rodriguez L.O. Hecht S.M. Haseltine W.A. J. Biol. Chem. 1983; 258: 711-713Abstract Full Text PDF PubMed Google Scholar), a one-base gap in each strand, and possible base in the Bennett O. J. Proc. Natl. Acad. Sci. U. S. A. 2000; PubMed Scopus Google Scholar). the complementarities for alignment of the during gap filling and ligation be for accurate restoration of the original sequence The requirement to repair of breaks by fragmentation on a defined or 3′ Chem. Res. 1992; PubMed Scopus Google Scholar, L.F. Mutat. Res. 1996; PubMed Scopus Google Scholar). The possibility that Ku be the was suggested by its to promote DNA (5Yaneva M. Kowalewski T. Lieber M.R. EMBO J. 1997; 16: 5098-5112Crossref PubMed Scopus (271) Google Scholar, 6Cary R.B. Peterson S.R. Wang J. Bear D.G. Bradbury E.M. Chen D.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4267-4272Crossref PubMed Scopus (228) Google Scholar, 7Pang D. Yoo S. Dynan W.S. Jung M. Dritschilo A. Cancer Res. 1997; 57: 1412-1415PubMed Google Scholar) and to the joining of either or cohesive DSBs by DNA M. EMBO J. PubMed Scopus Google Scholar, J. Mol. Biol. 2000; PubMed Scopus Google Scholar). The from of repair formed in CHO-K1 extracts during the of substrates with restriction ends (8Feldmann E. Schmiemann V. Goedecke W. Reichenberger S. Pfeiffer P. Nucleic Acids Res. 2000; 28: 2585-2596Crossref PubMed Scopus (191) Google Scholar). a for Ku in 3′-overhangs during end joining. (see to ligation, in were formed in extracts from The present results a for Ku in the accurate end joining of staggered free radical-mediated a break formed on a 3′ a and a gap in each a perhaps of the end joining in CHO-K1 extracts restoration of the original in Ku-deficient xrs6 extracts, accurate were end joining could be restored by the of purified D.W. Lees-Miller S.P. Biochem. Cell Biol. 1996; 74: PubMed Scopus Google Scholar) human Ku to the Ku for that have either or in Ku-deficient the in the fidelity of end joining. Ku in the of joining of a substrate with cohesive 5′-overhangs in this the requirement for Ku was not for the substrate the and the of joining in the was with that seen in CHO-K1 for the substrate the was by more than results suggest in the precise of Ku in the joining of the of breaks, that process is more to the hamster and human The of the substrates processing of the DNA ends to be at from of repair (8Feldmann E. Schmiemann V. Goedecke W. Reichenberger S. Pfeiffer P. Nucleic Acids Res. 2000; 28: 2585-2596Crossref PubMed Scopus (191) Google Scholar), processing and resection are more in Ku-deficient the of free for and ligation, at short not was in xrs6 and CHO-K1 extracts, of accurately in the xrs6 extracts was at than in CHO-K1 a requirement for Ku in alignment during gap filling and/or ligation, in to its in protecting ends from 3′ The enzyme that processing by the is not known, but is not the is the enzyme thus that is of has on 3′-overhangs D. Povirk L.F. Nucleic Acids Res. 1997; PubMed Scopus Google Scholar, B. M. Biochem. 1999; 57: PubMed Scopus Google Scholar). for from 3′ termini have been in extracts but have not been or M. Nucleic Acids Res. 1992; PubMed Scopus Google Scholar, T. B. Nucleic Acids Res. PubMed Scopus Google Scholar). from substrates with restriction ends (8Feldmann E. Schmiemann V. Goedecke W. Reichenberger S. Pfeiffer P. Nucleic Acids Res. 2000; 28: 2585-2596Crossref PubMed Scopus (191) Google Scholar), not in Ku or DNA-PK of DNA in and of microhomologies double strand (1Chu G. J. Biol. Chem. 1997; 272: 24097-24100Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar, 9Featherstone C. Jackson S.P. Mutat. Res. 1999; 434: 3-15Crossref PubMed Scopus (240) Google Scholar). the end joining consistent with were more in Ku-deficient xrs6 extracts and were by the of purified Ku in the Xenopus has been that joining is by of Ku and DNA-PKcs B. Reichenberger S. E. Pfeiffer P. J. Biochem. 258: PubMed Scopus Google Scholar). in vitro M. Proc. Natl. Acad. Sci. U. S. A. 2000; PubMed Scopus Google Scholar) suggest that the complex is a more for end joining based on microhomologies duplex DNA ends. extracts, Ku-deficient of accurate repair the DNA-PK on end joining in CHO-K1 results suggest that Ku and not DNA-PKcs is for accurate end joining in in human and Xenopus extracts, end joining of the substrates is but is the are to by the of that the for this is we of end joining by and DNA-PK in the in could be to the in of the extracts, could the of DNA-PK is typically in hamster and extracts than in human or Xenopus extracts T. Jackson EMBO J. 1985; PubMed Scopus Google Scholar, Lees-Miller S.P. 1992; Google Scholar, T. P.A. Jackson S.P. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, P. Mol. Biol. 1999; PubMed Scopus Google Scholar), and are consistent with this to a of DNA-PKcs protein in D.W. J. Lees-Miller S.P. 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Cancer Res. 1997; 57: 1412-1415PubMed Google Scholar). the present could be by a model of in vitro end joining is requirement for of Ku that the DNA ends in for and ligation to (see In and human extracts, Ku be from the ends and into the interior of DNA by DNA-PKcs, but not end has been proposed P. Frit P. Humbert O. Muller C. Chen D.J. Salles B. J. Biol. Chem. 1999; 274: 7848-7856Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar), DNA-PKcs, or protein in the promote either DNA-PKcs dissociation or of the complex the DNA thus allowing Ku to to its the ends. of and during end joining has been in Xenopus extracts P. Mol. Biol. 1999; PubMed Scopus Google Scholar), in we have Chen and F. in this the of DNA-PKcs is Ku and thus the of end processing the of to be The of DNA-PKcs in accurate end joining in CHO-K1 extracts not that in end joining in at lacking DNA-PKcs the and repair lacking Ku P.A. Mutat. Res. 1997; 384: 1-14Crossref PubMed Scopus (161) Google Scholar). possible for this is that DNA-PKcs a in DNA ends a that be more and complex in than in in vitro free ends are with this DNA-PKcs has been to promote ligation by ligation P. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). the that end joining extracts but not at in human extracts, of DNA-PKcs in T. Jackson EMBO J. 1985; PubMed Scopus Google Scholar, Lees-Miller S.P. 1992; Google Scholar, T. P.A. Jackson S.P. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, P. Mol. Biol. 1999; PubMed Scopus Google Scholar), to be in suggest that DNA-PK be or at for the of that of V. M. Curr. Biol. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). of at repair the of and/or the complex at the that repair to sites of or DNA-PK a in a complex that to repair by ends to repair J. Genet. 1999; 64: Full Text Full Text PDF PubMed Scopus (161) Google Scholar, J.F. PubMed Scopus Google Scholar, Mol. Biol. 1997; PubMed Scopus Google Scholar), of not Ku for end a in DNA-PK than in end alignment could be the for the of The of a the of Ku in each of functions and thus of their in repair repair and The be in and biochemical of and are for in the end joining for and for a of the

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