Non-homologous End Joining Requires That the DNA-PK Complex Undergo an Autophosphorylation-dependent Rearrangement at DNA Ends

Repair of chromosome breaks by non-homologous end joining requires the XRCC4-ligase IV complex, Ku, and the DNA-dependent protein kinase catalytic subunit (DNA-PKcs). DNA-PKcs must also retain kinase activity and undergo autophosphorylation at six closely linked sites (ABCDE sites). We describe here an end-joining assay using only purified components that reflects cellular requirements for both Ku and kinase-active DNA-PKcs and investigate the mechanistic basis for these requirements. A need for DNA-PKcs autophosphorylation is sufficient to explain the requirement for kinase activity, in part because autophosphorylation is generally required for end-joining factors to access DNA ends. However, DNA-PKcs with all six ABCDE autophosphorylation sites mutated to alanine allows access to ends through autophosphorylation of other sites, yet our in vitro end-joining assay still reflects the defectiveness of this mutant in cellular end joining. In contrast, mutation of ABCDE sites to aspartate, a phosphorylation mimic, supports high levels of end joining that is now independent of kinase activity. This is likely because DNA-PKcs with aspartate substitutions at ABCDE sites allow access to DNA ends while retaining affinity for Ku-bound ends and stabilizing recruitment of the XRCC4-ligase IV complex. Autophosphorylation at ABCDE sites thus apparently directs a rearrangement of the DNA-PK complex that ensures access to broken ends and joining steps are coupled together within a synaptic complex, making repair more accurate. Repair of chromosome breaks by non-homologous end joining requires the XRCC4-ligase IV complex, Ku, and the DNA-dependent protein kinase catalytic subunit (DNA-PKcs). DNA-PKcs must also retain kinase activity and undergo autophosphorylation at six closely linked sites (ABCDE sites). We describe here an end-joining assay using only purified components that reflects cellular requirements for both Ku and kinase-active DNA-PKcs and investigate the mechanistic basis for these requirements. A need for DNA-PKcs autophosphorylation is sufficient to explain the requirement for kinase activity, in part because autophosphorylation is generally required for end-joining factors to access DNA ends. However, DNA-PKcs with all six ABCDE autophosphorylation sites mutated to alanine allows access to ends through autophosphorylation of other sites, yet our in vitro end-joining assay still reflects the defectiveness of this mutant in cellular end joining. In contrast, mutation of ABCDE sites to aspartate, a phosphorylation mimic, supports high levels of end joining that is now independent of kinase activity. This is likely because DNA-PKcs with aspartate substitutions at ABCDE sites allow access to DNA ends while retaining affinity for Ku-bound ends and stabilizing recruitment of the XRCC4-ligase IV complex. Autophosphorylation at ABCDE sites thus apparently directs a rearrangement of the DNA-PK complex that ensures access to broken ends and joining steps are coupled together within a synaptic complex, making repair more accurate. The mammalian non-homologous end-joining pathway has an important role in all cell types for repair of double strand breaks (DSBs) 1The abbreviations used are: DSB, double strand break; LX, XRCC4-ligase IV complex; PKcs, protein kinase catalytic subunit; DNA-PK, DNA-dependent protein kinase. caused by DNA damage (e.g. after ionizing radiation). End joining is also essential for efficient resolution of DSB intermediates in V(D)J recombination, a lymphocytespecific process required for assembly of the antigen-specific receptors of the immune system. Defective end joining thus results in radiation sensitivity, an increased incidence of cancer, and severe immunodeficiency (1Gellert M. Annu. Rev. Biochem. 2002; 71: 101-132Crossref PubMed Scopus (621) Google Scholar). Repair of broken ends by this pathway requires 1) recognition of broken ends and recruitment of end-joining factors, 2) alignment or synapsis of a pair of ends, and 3) processing by polymerases or nucleases as needed to generate compatible ends 4) followed by ligation. End joining uses the XRCC4-ligase IV complex (LX) for the ligation step (2Wilson T.E. Grawunder U. Lieber M.R. Nature. 1997; 388: 495-498Crossref PubMed Scopus (349) Google Scholar, 3Grawunder U. Wilm M. Wu X. Kulesza P. Wilson T.E. Mann M. Lieber M.R. Nature. 1997; 388: 492-495Crossref PubMed Scopus (546) Google Scholar), whereas the processing step uses at least the Artemis nuclease (4Ma Y. Pannicke U. Schwarz K. Lieber M.R. Cell. 2002; 108: 781-794Abstract Full Text Full Text PDF PubMed Scopus (851) Google Scholar, 5Rooney S. Sekiguchi J. Zhu C. Cheng H.L. Manis J. Whitlow S. DeVido J. Foy D. Chaudhuri J. Lombard D. Alt F.W. Mol. Cell. 2002; 10: 1379-1390Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar) and polymerase μ (6Mahajan K.N. Nick McElhinny S.A. Mitchell B.S. Ramsden D.A. Mol. Cell. Biol. 2002; 22: 5194-5202Crossref PubMed Scopus (249) Google Scholar, 7Bertocci B. De Smet A. Berek C. Weill J.C. Reynaud C.A. Immunity. 2003; 19: 203-211Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). The DNA-dependent protein kinase (DNA-PK) has been linked to early steps in end joining, and consists of the Ku heterodimer (Ku70 and Ku80) and a 460-kDa serine/threonine DNA-PK catalytic subunit (DNA-PKcs). Ku is required for end recognition and, once loaded on ends, acts as a scaffold for subsequent recruitment of DNA-PKcs (8Gottlieb T.M. Jackson S.P. Cell. 1993; 72: 131-142Abstract Full Text PDF PubMed Scopus (1030) Google Scholar), the LX complex (9Nick McElhinny S.A. Snowden C.M. McCarville J. Ramsden D.A. Mol. Cell. Biol. 2000; 20: 2996-3003Crossref PubMed Scopus (319) Google Scholar, 10Chen L. Trujillo K. Sung P. Tomkinson A.E. J. Biol. Chem. 2000; 275: 26196-26205Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar, 11Teo S.H. Jackson S.P. Curr. Biol. 2000; 10: 165-168Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar), polymerase μ (6Mahajan K.N. Nick McElhinny S.A. Mitchell B.S. Ramsden D.A. Mol. Cell. Biol. 2002; 22: 5194-5202Crossref PubMed Scopus (249) Google Scholar), and probably other factors as well. The role of DNA-PKcs is not well understood. Several observations, including its ability to associate with Artemis and activate the endonuclease activity of Artemis (4Ma Y. Pannicke U. Schwarz K. Lieber M.R. Cell. 2002; 108: 781-794Abstract Full Text Full Text PDF PubMed Scopus (851) Google Scholar), suggest a specific role for DNA-PKcs in directing end-processing events; however, DNA-PKcs deficiency affects cellular end joining even when processing is not required (see Ref. 12Bogue M.A. Jhappan C. Roth D.B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 15559-15564Crossref PubMed Scopus (78) Google Scholar and references therein). A more general role for DNA-PKcs in end joining is also consistent with in vitro evidence indicating that it helps Ku recruit the LX complex to ends (10Chen L. Trujillo K. Sung P. Tomkinson A.E. J. Biol. Chem. 2000; 275: 26196-26205Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar, 13Calsou P. Delteil C. Frit P. Drouet J. Salles B. J. Mol. Biol. 2003; 326: 93-103Crossref PubMed Scopus (98) Google Scholar) and promotes the intermolecular synapsis of two DNA ends (14DeFazio L.G. Stansel R.M. Griffith J.D. Chu G. EMBO J. 2002; 21: 3192-3200Crossref PubMed Scopus (270) Google Scholar). Together, these functions of DNA-PKcs may explain why LX activity is necessary in vitro when using substrates that do not require processing (10Chen L. Trujillo K. Sung P. Tomkinson A.E. J. Biol. Chem. 2000; 275: 26196-26205Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar, 15Hanakahi L.A. Bartlet-Jones M. Chappell C. Pappin D. West S.C. Cell. 2000; 102: 721-729Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar). Recent studies indicate that cellular end joining requires DNA-PKcs to undergo autophosphorylation (16Soubeyrand S. Pope L. Pakuts B. Hache R.J. Cancer Res. 2003; 63: 1198-1201PubMed Google Scholar, 17Ding Q. Reddy Y.V. Wang W. Woods T. Douglas P. Ramsden D.A. Lees-Miller S.P. Meek K. Mol. Cell. Biol. 2003; 23: 5836-5848Crossref PubMed Scopus (264) Google Scholar, 18Chan D.W. Chen B.P. Prithivirajsingh S. Kurimasa A. Story M.D. Qin J. Chen D.J. Genes Dev. 2002; 16: 2333-2338Crossref PubMed Scopus (389) Google Scholar). Six autophosphorylation sites are clustered within a 40-amino acid region (“ABCDE” sites) (see Fig. 2A), although at least two other sites are also autophosphorylated (19Douglas P. Sapkota G.P. Morrice N. Yu Y. Goodarzi A.A. Merkle D. Meek K. Alessi D.R. Lees-Miller S.P. Biochem. J. 2002; 368: 243-251Crossref PubMed Scopus (161) Google Scholar). Individual mutation of ABCDE sites has mild or negligible effects, but a mutant with all six ABCDE sites converted to alanine (ABCDE(ala)) is unable to complement DNA-PKcs-deficient cells (17Ding Q. Reddy Y.V. Wang W. Woods T. Douglas P. Ramsden D.A. Lees-Miller S.P. Meek K. Mol. Cell. Biol. 2003; 23: 5836-5848Crossref PubMed Scopus (264) Google Scholar). Autophosphorylation in general had previously been shown in vitro to trigger a dissociation of DNA-PKcs from Ku-bound ends (20Merkle D. Douglas P. Moorhead G.B. Leonenko Z. Yu Y. Cramb D. Bazett-Jones D.P. Lees-Miller S.P. Biochemistry. 2002; 41: 12706-12714Crossref PubMed Scopus (131) Google Scholar, 21Chan D.W. Lees-Miller S.P. J. Biol. Chem. 1996; 271: 8936-8941Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar) and is required to make ends accessible to exogenous factors (e.g. exonucleases) (22Weterings E. Verkaik N.S. Bruggenwirth H.T. Hoeijmakers J.H. van Gent D.C. Nucleic Acids Res. 2003; 31: 7238-7246Crossref PubMed Scopus (103) Google Scholar, 23Calsou 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 (91) Google Scholar) and probably end-joining factors as well (24Udayakumar D. Bladen C.L. Hudson F.Z. Dynan W.S. J. Biol. Chem. 2003; 278: 41631-41635Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). Together, this evidence suggests that the cellular defects of the ABCDE(ala) mutant could simply be explained by its inability to be autophosphorylated and then dissociate, because its continued presence would prevent access to DNA ends by other end-joining factors. However, the purified ABCDE(ala) mutant protein also dissociates after autophosphorylation (17Ding Q. Reddy Y.V. Wang W. Woods T. Douglas P. Ramsden D.A. Lees-Miller S.P. Meek K. Mol. Cell. Biol. 2003; 23: 5836-5848Crossref PubMed Scopus (264) Google Scholar). Autophosphorylation of sites outside of the ABCDE cluster is therefore sufficient for dissociation and end access, and the mechanism by which ABCDE autophosphorylation promotes end joining remains unclear. We therefore developed an in vitro assay using only purified Ku, DNA-PKcs, and the LX complex that reflects the cellular requirement for autophosphorylation at the ABCDE sites. We show that autophosphorylation at ABCDE sites uniquely makes ends accessible while still allowing DNA-PKcs to persist at ends and retain LX within a complex at ends. Proteins—Recombinant Ku heterodimer and LX were purified from baculovirus-infected cells as described previously (9Nick McElhinny S.A. Snowden C.M. McCarville J. Ramsden D.A. Mol. Cell. Biol. 2000; 20: 2996-3003Crossref PubMed Scopus (319) Google Scholar). DNA-PKcs was purified from 50 g of human placenta (“native” DNA-PKcs) or 5 liters of V3 cells stably expressing the various constructs (recombinant), also as described previously (17Ding Q. Reddy Y.V. Wang W. Woods T. Douglas P. Ramsden D.A. Lees-Miller S.P. Meek K. Mol. Cell. Biol. 2003; 23: 5836-5848Crossref PubMed Scopus (264) Google Scholar), with the following exception. Instead of using polyethyleneimine cellulose, nucleic acids were depleted by addition of polyethyleneimine to the extract to 0.2% v/v, centrifugation at 15,000 × g for 15 min, and further clarification of the extract by the addition of phosphocellulose to 10% v/v and filtration. Approximate concentrations of proteins were calculated by Bradford assay using bovine serum as a and used to these were within when kinase activity of DNA-PKcs was by of a protein with in for on and activity of DNA-PKcs with DNA-PKcs in the with the of 50 bovine serum and used a of LX activity with this required Ku as as LX was not in DNA ends. requirement for DNA-PKcs was when the of Ku also not the of DNA ends and when concentrations of all including DNA ends, were were with 10% and, as for Fig. with Ku and DNA-PKcs for at the addition of LX, 5 and and to Fig. to at the a with and but shown in Fig. were then by the addition of LX and to were by the addition of an of a 5 0.2% and at for and with an of and on a were by DNA with and by using the as a The of was using a and The DNA used in has been described previously (9Nick McElhinny S.A. Snowden C.M. McCarville J. Ramsden D.A. Mol. Cell. Biol. 2000; 20: 2996-3003Crossref PubMed Scopus (319) Google Scholar). were in and for at with with was not required for of but this the of were on a in and at used the described and of This was by of to be the of necessary to of the in 5 in the of other were by at for 5 min, after which 5 and were were then at and at the also as described for ligation were to on an and were with DNA for DNA-PK in in End the role of Ku and DNA-PKcs in in vitro end joining by the LX complex using a DNA with DNA-PK are required for LX activity when using the described here also end joining is more when the kinase activity of DNA-PKcs is by with 5 and We autophosphorylation of DNA-PK was sufficient to explain the requirement for kinase activity by the autophosphorylation and ligation DNA-PK was autophosphorylated to in the by addition of at the after or after of In the LX was to well autophosphorylated DNA-PKcs end joining but now kinase activity. a for autophosphorylation and ligation to in as in Fig. by and and the addition of LX of autophosphorylation was sufficient to make DNA-PKcs independent of kinase activity in the end-joining assay and Autophosphorylation of DNA-PK is thus sufficient to explain the role of kinase activity in this and autophosphorylation need not in with end joining to be autophosphorylation still end-joining activity, but levels were to the autophosphorylation and although at least autophosphorylation is required for end joining, the autophosphorylation after is We purified DNA-PKcs with at sites of autophosphorylation A and and activity in this assay ligation activity to DNA-PKcs is when using a mutant in which autophosphorylation of ABCDE and is by alanine substitutions (ABCDE(ala)) and results were with independent of ABCDE(ala) and also after a phosphorylation outside the ABCDE cluster was (19Douglas P. Sapkota G.P. Morrice N. Yu Y. Goodarzi A.A. Merkle D. Meek K. Alessi D.R. Lees-Miller S.P. Biochem. J. 2002; 368: 243-251Crossref PubMed Scopus (161) Google Scholar). of this in with ABCDE sites also to both in cellular Meek and Q. and in vitro In contrast, mutation of sites in the ABCDE cluster not end joining in vitro Reddy and D. A. consistent with the ability of these to or V(D)J in cells (17Ding Q. Reddy Y.V. Wang W. Woods T. Douglas P. Ramsden D.A. Lees-Miller S.P. Meek K. Mol. Cell. Biol. 2003; 23: 5836-5848Crossref PubMed Scopus (264) Google Scholar). The requirements of this in vitro end-joining assay for autophosphorylation within ABCDE to requirements previously in cellular end-joining (17Ding Q. Reddy Y.V. Wang W. Woods T. Douglas P. Ramsden D.A. Lees-Miller S.P. Meek K. Mol. Cell. Biol. 2003; 23: 5836-5848Crossref PubMed Scopus (264) Google Scholar). of ABCDE of end joining with the ABCDE(ala) mutant are to when kinase activity of DNA-PKcs is indicating that autophosphorylation of ABCDE sites is sufficient to explain the role of kinase activity in this However, ABCDE(ala) is from DNA-PKcs in other This mutant levels of kinase activity as using Artemis (17Ding Q. Reddy Y.V. Wang W. Woods T. Douglas P. Ramsden D.A. Lees-Miller S.P. Meek K. Mol. Cell. Biol. 2003; 23: 5836-5848Crossref PubMed Scopus (264) Google Scholar), or Ku as Meek and Q. also that ABCDE(ala) has affinity for Ku-bound DNA ends (17Ding Q. Reddy Y.V. Wang W. Woods T. Douglas P. Ramsden D.A. Lees-Miller S.P. Meek K. Mol. Cell. Biol. 2003; 23: 5836-5848Crossref PubMed Scopus (264) Google Scholar) and and the ability of ABCDE(ala) to recruitment of LX to DNA is to DNA-PKcs and ABCDE(ala) not as from Ku-bound ends as DNA-PKcs after but it to (17Ding Q. Reddy Y.V. Wang W. Woods T. Douglas P. Ramsden D.A. Lees-Miller S.P. Meek K. Mol. Cell. Biol. 2003; 23: 5836-5848Crossref PubMed Scopus (264) Google Scholar) Autophosphorylation of sites outside the ABCDE cluster is thus sufficient for dissociation of We used the ABCDE(ala) mutant and an assay to autophosphorylation of sites outside the ABCDE cluster is sufficient to make DNA ends accessible Ku and DNA-PKcs are both required to by when both factors are DNA is well only when the kinase is consistent with studies indicating that autophosphorylation is required for to access DNA ends (22Weterings E. Verkaik N.S. Bruggenwirth H.T. Hoeijmakers J.H. van Gent D.C. Nucleic Acids Res. 2003; 31: 7238-7246Crossref PubMed Scopus (103) Google Scholar, 23Calsou 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 (91) Google Scholar) with the ABCDE(ala) mutant is from DNA-PKcs in this assay as because autophosphorylation of protein to the of by with DNA-PKcs and ABCDE(ala) thus both allow access to ends on but only DNA-PKcs supports high levels of joining by the LX complex. We that simply access to ends not explain why autophosphorylation at ABCDE sites is for cellular end joining. We suggest that ABCDE autophosphorylation may be essential for end-joining activity because it end while still allowing DNA-PKcs to of DNA-PKcs at ends would allow it to synapsis of ends and stabilizing recruitment of LX, thus LX to of end This that DNA-PKcs autophosphorylated only within ABCDE sites allow access to ends but retain the ability to a complex with end-joining factors. these a DNA-PKcs in which autophosphorylation is to ABCDE sites by using a mutant with phosphorylation at ABCDE sites of In the presence of ends are to even when kinase activity is by that autophosphorylation to ABCDE is sufficient for end still a complex with Ku at DNA ends, although as well as DNA-PKcs and kinase activity (17Ding Q. Reddy Y.V. Wang W. Woods T. Douglas P. Ramsden D.A. Lees-Miller S.P. Meek K. Mol. Cell. Biol. 2003; 23: 5836-5848Crossref PubMed Scopus (264) Google Scholar), and autophosphorylation of this mutant at sites outside the ABCDE cluster is required for dissociation 5 and as previously (17Ding Q. Reddy Y.V. Wang W. Woods T. Douglas P. Ramsden D.A. Lees-Miller S.P. Meek K. Mol. Cell. Biol. 2003; 23: 5836-5848Crossref PubMed Scopus (264) Google Scholar) from the of the ABCDE(ala) still promotes recruitment of the LX complex with and DNA-PKcs autophosphorylated only within ABCDE thus access to ends, affinity for Ku-bound ends, and helps retain LX at ends. We shown that at least autophosphorylation of DNA-PKcs within ABCDE sites is essential for end-joining activity and but the described suggest it may also be sufficient autophosphorylation outside ABCDE may be for end-joining activity in this We this by was in the end-joining even when kinase activity was by was only DNA-PKcs in end joining, in to ABCDE(ala) and the ability of DNA-PKcs to end joining requires kinase activity, but not and This that autophosphorylation within ABCDE is both necessary and sufficient for activity of LX in We developed an in vitro end-joining assay using only purified Ku, DNA-PKcs, and are the factors required for non-homologous end joining in that our assay reflects the cellular end-joining our on the ability of DNA-PKcs to undergo autophosphorylation at ABCDE sites in to the in results using this assay indicate that ABCDE autophosphorylation to a of DNA-PK that is for end-joining activity. autophosphorylation outside the ABCDE cluster has a on DNA-PK complex but not end joining. We therefore that autophosphorylation within ABCDE is uniquely to a rearrangement of the DNA-PK complex at DNA ends that is essential for end joining both in vitro and in This requirement important to the end joining repair pathway because it ensures the of step in end joining to the as described Ku and DNA-PKcs recruit LX and other end-joining factors to DNA ends, but ends even to factors within this complex when autophosphorylation is (22Weterings E. Verkaik N.S. Bruggenwirth H.T. Hoeijmakers J.H. van Gent D.C. Nucleic Acids Res. 2003; 31: 7238-7246Crossref PubMed Scopus (103) Google Scholar, 23Calsou 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 (91) Google Scholar, D. Bladen C.L. Hudson F.Z. Dynan W.S. J. Biol. Chem. 2003; 278: 41631-41635Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). Autophosphorylation kinase activity in is when ends are together step 2) (14DeFazio L.G. Stansel R.M. Griffith J.D. Chu G. EMBO J. 2002; 21: 3192-3200Crossref PubMed Scopus (270) Google Scholar, E. Verkaik N.S. Bruggenwirth H.T. Hoeijmakers J.H. van Gent D.C. Nucleic Acids Res. 2003; 31: 7238-7246Crossref PubMed Scopus (103) Google Scholar). Autophosphorylation thus end to to that only specific to this and thus within the synaptic complex, on ends. The mutant from This DNA-PKcs has only levels of end joining to and the ABCDE(ala) both in vitro and in cellular (17Ding Q. Reddy Y.V. Wang W. Woods T. Douglas P. Ramsden D.A. Lees-Miller S.P. Meek K. Mol. Cell. Biol. 2003; 23: 5836-5848Crossref PubMed Scopus (264) Google Scholar). However, of the mutant is in probably of the by end in a cellular the kinase is autophosphorylation both within the ABCDE cluster and at other sites that are indicate that ABCDE and other sites are autophosphorylated and of is sufficient to make ends accessible that the with cells expressing the ABCDE(ala) mutant is not that this mutant the ability of processing factors to access ends. In of this intermediates in V(D)J are with in cells expressing DNA-PKcs or the ABCDE(ala) but these intermediates are not in cells expressing ABCDE(ala) (17Ding Q. Reddy Y.V. Wang W. Woods T. Douglas P. Ramsden D.A. Lees-Miller S.P. Meek K. Mol. Cell. Biol. 2003; 23: 5836-5848Crossref PubMed Scopus (264) Google Scholar). the ABCDE(ala) mutant is also unable to levels of end joining in a that not require processing (17Ding Q. Reddy Y.V. Wang W. Woods T. Douglas P. Ramsden D.A. Lees-Miller S.P. Meek K. Mol. Cell. Biol. 2003; 23: 5836-5848Crossref PubMed Scopus (264) Google Scholar). The with end joining in cells expressing ABCDE(ala) must therefore be that are unable to synapsis with end access and LX activity probably the activity of other end-joining factors as This is because ABCDE autophosphorylation of DNA-PKcs is uniquely to end with its at ends step to ends in this DNA-PKcs its role in end synapsis and the recruitment of LX repair is Autophosphorylation to ABCDE sites or outside sites has an on well DNA-PKcs with Ku-bound DNA ends with and However, our suggest that autophosphorylation within ABCDE allows access to ends to DNA-PKcs whereas autophosphorylation of outside sites access only when a sufficient of sites been that DNA-PKcs This suggests a mechanistic in the two types of autophosphorylation DNA is likely that ABCDE autophosphorylation the DNA-PK complex by its on the DNA activity of DNA-PKcs to the specific of in end step In contrast, autophosphorylation at sites outside the ABCDE cluster an not in end or DNA by with with the and a for DNA-PKcs with in which with DNA ends, whereas the other with the DNA from the end J. A. J.D. P. O. EMBO J. 2003; 22: PubMed Scopus Google Scholar). autophosphorylation of DNA-PKcs to a in activity and dissociation step 4) (20Merkle D. Douglas P. Moorhead G.B. Leonenko Z. Yu Y. Cramb D. Bazett-Jones D.P. Lees-Miller S.P. Biochemistry. 2002; 41: 12706-12714Crossref PubMed Scopus (131) Google Scholar, 21Chan D.W. Lees-Miller S.P. J. Biol. Chem. 1996; 271: 8936-8941Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar). This may be required for processing that require a of access to ends ligation. because DNA-PKcs has a role in stabilizing recruitment of LX (10Chen L. Trujillo K. Sung P. Tomkinson A.E. J. Biol. Chem. 2000; 275: 26196-26205Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar, 13Calsou P. Delteil C. Frit P. Drouet J. Salles B. J. Mol. Biol. 2003; 326: 93-103Crossref PubMed Scopus (98) Google Scholar), Artemis (4Ma Y. Pannicke U. Schwarz K. Lieber M.R. Cell. 2002; 108: 781-794Abstract Full Text Full Text PDF PubMed Scopus (851) Google Scholar), and likely other factors as dissociation of DNA-PKcs may trigger the general of the end-joining complex, with the of of the end-joining complex could be important in making the accessible for subsequent (e.g. and the of end-joining factors from breaks when repair may cells by making these breaks accessible to other repair The is by the ability of a specific of DNA-PKcs kinase activity to repair of C. J. Mol. Cancer Res. 2003; Google Scholar). still be by end joining in the of DNA-PK and but this pathway is M. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar, L. Roth D.B. Nucleic Acids Res. 1998; PubMed Scopus Google Scholar, N.S. van D. Bruggenwirth H.T. Hoeijmakers J.H. van Gent D.C. J. 2002; PubMed Scopus Google Scholar) and more to C. C. Manis J. J. Y. Alt F.W. Cell. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar) when DNA-PK is We suggest that a for this is the ability of ABCDE autophosphorylation to that once ends been by DNA-PK only the factors to this and thus with DNA-PK in the synaptic complex, be the other processing that are not coupled to synapsis be DNA-PK makes end joining more accurate. We Nick E. and for of the and of the Ramsden for

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