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• W2119371129 abstract "Cernunnos-XLF is the most recently identified core component in the nonhomologous end-joining (NHEJ) pathway for the repair of DNA double strand breaks (DSBs) in mammals. It associates with the XRCC4/ligase IV ligation complex and stimulates its activity in a still unknown manner. NHEJ also requires the DNA-dependent protein kinase that contains a Ku70/Ku80 heterodimer and the DNA-dependent protein kinase catalytic subunit. To understand the interplay between Cernunnos-XLF and the other proteins implicated in the NHEJ process, we have analyzed the interactions of Cernunnos-XLF and NHEJ proteins in cells after treatment with DNA double strand-breaking agents by means of a detergent-based cellular fractionation protocol. We report that Cernunnos-XLF is corecruited with the core NHEJ components on chromatin damaged with DSBs in human cells and is phosphorylated by the DNA-dependent protein kinase catalytic subunit. Our data show a pivotal role for DNA ligase IV in the NHEJ ligation complex assembly and recruitment to DSBs because the association of Cernunnos-XLF with the XRCC4/ligase IV complex relies primarily on the DNA ligase IV component, and an intact XRCC4/ligase IV complex is necessary for Cernunnos-XLF mobilization to damaged chromatin. Conversely, a Cernunnos-XLF defect has no apparent impact on the XRCC4/ligase IV association and recruitment to the DSBs or on the stimulation of the DNA-dependent protein kinase on DNA ends. Cernunnos-XLF is the most recently identified core component in the nonhomologous end-joining (NHEJ) pathway for the repair of DNA double strand breaks (DSBs) in mammals. It associates with the XRCC4/ligase IV ligation complex and stimulates its activity in a still unknown manner. NHEJ also requires the DNA-dependent protein kinase that contains a Ku70/Ku80 heterodimer and the DNA-dependent protein kinase catalytic subunit. To understand the interplay between Cernunnos-XLF and the other proteins implicated in the NHEJ process, we have analyzed the interactions of Cernunnos-XLF and NHEJ proteins in cells after treatment with DNA double strand-breaking agents by means of a detergent-based cellular fractionation protocol. We report that Cernunnos-XLF is corecruited with the core NHEJ components on chromatin damaged with DSBs in human cells and is phosphorylated by the DNA-dependent protein kinase catalytic subunit. Our data show a pivotal role for DNA ligase IV in the NHEJ ligation complex assembly and recruitment to DSBs because the association of Cernunnos-XLF with the XRCC4/ligase IV complex relies primarily on the DNA ligase IV component, and an intact XRCC4/ligase IV complex is necessary for Cernunnos-XLF mobilization to damaged chromatin. Conversely, a Cernunnos-XLF defect has no apparent impact on the XRCC4/ligase IV association and recruitment to the DSBs or on the stimulation of the DNA-dependent protein kinase on DNA ends. DNA double strand breaks (DSBs) 3The abbreviations used are: DSB, double strand break; NHEJ, nonhomologous end-joining; DNA-PK, DNA-dependent protein kinase; DNA-PKcs, DNA-dependent protein kinase catalytic subunit; IR, ionizing radiation; WCE, whole cell extract; PBS, phosphate-buffered saline; Cali, calicheamicin γ1; shRNA, short hairpin RNA; shPKcs, short hairpin protein kinase catalytic subunit.3The abbreviations used are: DSB, double strand break; NHEJ, nonhomologous end-joining; DNA-PK, DNA-dependent protein kinase; DNA-PKcs, DNA-dependent protein kinase catalytic subunit; IR, ionizing radiation; WCE, whole cell extract; PBS, phosphate-buffered saline; Cali, calicheamicin γ1; shRNA, short hairpin RNA; shPKcs, short hairpin protein kinase catalytic subunit. occur physiologically in lymphocytes during V(D) J recombination or class-switch recombination (1Dudley D.D. Kim J. Bassing C.H. Alt F.W. Adv. Immunol. 2005; 86: 43-112Crossref PubMed Scopus (212) Google Scholar). However, DSBs in most cells are pathologic like those arising from genomic replication in the presence of nicks or from cell treatment with ionizing radiation (IR) or radiomimetic molecules. Improper repair of DSBs may lead to cell death or cancer-prone genomic rearrangements (2Lieber M.R. Kim K. Raghavan S.C. DNA Repair (Amst.). 2006; 5: 1234-1245Crossref PubMed Scopus (149) Google Scholar, 3O'Driscoll M. Kim P.A. Nat. Rev. Genet. 2006; 7: 45-54Crossref PubMed Scopus (452) Google Scholar).In mammalian cells, apart from homologous recombination between sister chromatids, DSBs are mainly processed by in situ religation relying on the nonhomologous end-joining (NHEJ) pathway (4Burma S. Kim B.P. Chen D.J. DNA Repair (Amst.). 2006; 5: 1042-1048Crossref PubMed Scopus (301) Google Scholar). Although alternative subpathways may operate (5Audebert M. Kim B. Calsou P. J. Biol. Chem. 2004; 279: 55117-55126Abstract Full Text Full Text PDF PubMed Scopus (538) Google Scholar, 6Wang M. Kim W. Wu W. Rosidi B. Zhang L. Wang H. Iliakis G. Nucleic Acids Res. 2006; 34: 6170-6182Crossref PubMed Scopus (585) Google Scholar), the major NHEJ pathway relies on a set of core proteins, the individual deficiency of which elicits a radiosensitive severe combined immunodeficiency syndrome in human or animals (7de Villartay J.P. Kim C. de Chasseval R. Buck D. Le Guyader G. Villey I. Curr. Opin. Immunol. 2003; 15: 592-598Crossref PubMed Scopus (23) Google Scholar). The two DNA ends of the DSB are recognized and bound by the ring-shaped heterodimer Ku70/Ku80 that recruits the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) (8Downs J.A. Kim S.P. Nat. Rev. Mol. Cell Biol. 2004; 5: 367-378Crossref PubMed Scopus (301) Google Scholar). The assembled DNA-dependent protein kinase (DNA-PK) holoenzyme then exhibits serine-threonine protein kinase and DNA end-bridging activities (9Collis S.J. Kim T.L. Jeggo P.A. Parker A.R. Oncogene. 2005; 24: 949-961Crossref PubMed Scopus (370) Google Scholar, 10Meek K. Kim S. Ramsden D.A. Lees-Miller S.P. Immunol. Rev. 2004; 200: 132-141Crossref PubMed Scopus (176) Google Scholar). Among other functions, the kinase activity regulates DNA end access to processing enzymes like the DNA-PKcs-associated Artemis nuclease (11Drouet J. Kim P. Delteil C. de Villartay J.P. Salles B. Calsou P. J. Biol. Chem. 2006; 281: 27784-27793Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 12Goodarzi A.A. Kim Y. Riballo E. Douglas P. Walker S.A. Ye R. Harer C. Marchetti C. Morrice N. Jeggo P.A. Lees-Miller S.P. EMBO J. 2006; 25: 3880-3889Crossref PubMed Scopus (225) Google Scholar, 13Niewolik D. Kim U. Lu H. Ma Y. Wang L.C. Kulesza P. Zandi E. Lieber M.R. Schwarz K. J. Biol. Chem. 2006; 281: 33900-33909Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). This explains why DNA-PK is a favored target in radiosensitization strategies of tumors (14Salles B. Kim P. Frit P. Muller C. Pathol. Biol. 2006; 54: 185-193Crossref PubMed Scopus (57) Google Scholar). Finally, the XRCC4/DNA ligase IV complex is responsible for the ligation step (15Grawunder U. Kim D. Fugmann S. Schwarz K. Lieber M.R. Mol. Cell. 1998; 2: 477-484Abstract Full Text Full Text PDF PubMed Scopus (282) Google Scholar, 16Li Z. Kim T. Gao Y. Cheng H.L. Seed B. Stamato T.D. Taccioli G.E. Alt F.W. Cell. 1995; 83: 1079-1089Abstract Full Text PDF PubMed Scopus (397) Google Scholar).Another core NHEJ factor is Cernunnos-XLF, a factor with a predicted structural similarity to XRCC4 that has been identified as an XRCC4-interacting protein (18Ahnesorg P. Kim P. Jackson S.P. Cell. 2006; 124: 301-313Abstract Full Text Full Text PDF PubMed Scopus (582) Google Scholar) that is deficient in a human radiosensitive severe combined immunodeficiency syndrome (17Buck D. Kim L. de Chasseval R. Barraud A. Fondaneche M.C. Sanal O. Plebani A. Stephan J.L. Hufnagel M. le Deist F. Fischer A. Durandy A. de Villartay J.P. Revy P. Cell. 2006; 124: 287-299Abstract Full Text Full Text PDF PubMed Scopus (579) Google Scholar). Cernunnos-XLF is the homologue of the yeast protein Nej1p in Saccharomyces cerevisiae (19Callebaut I. Kim L. Fischer A. Mornon J.P. Revy P. de Villartay J.P. J. Biol. Chem. 2006; 281: 13857-13860Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar) and belongs to a larger family of functionally conserved proteins that are required for NHEJ (20Hentges P. Kim P. Pitcher R.S. Bruce C.K. Kysela B. Green A.J. Bianchi J. Wilson T.E. Jackson S.P. Doherty A.J. J. Biol. Chem. 2006; 281: 37517-37526Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). This factor has been postulated to function in NHEJ events based on its interaction with the XRCC4/ligase IV complex (18Ahnesorg P. Kim P. Jackson S.P. Cell. 2006; 124: 301-313Abstract Full Text Full Text PDF PubMed Scopus (582) Google Scholar, 19Callebaut I. Kim L. Fischer A. Mornon J.P. Revy P. de Villartay J.P. J. Biol. Chem. 2006; 281: 13857-13860Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 21Lu H. Kim U. Schwarz K. Lieber M.R. J. Biol. Chem. 2007; 282: 11155-11162Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar), the lack of V(D) J recombination activity in plasmid transfection assays and on the high IR sensitivity of the corresponding deficient cells (17Buck D. Kim L. de Chasseval R. Barraud A. Fondaneche M.C. Sanal O. Plebani A. Stephan J.L. Hufnagel M. le Deist F. Fischer A. Durandy A. de Villartay J.P. Revy P. Cell. 2006; 124: 287-299Abstract Full Text Full Text PDF PubMed Scopus (579) Google Scholar, 18Ahnesorg P. Kim P. Jackson S.P. Cell. 2006; 124: 301-313Abstract Full Text Full Text PDF PubMed Scopus (582) Google Scholar, 22Zha S. Kim F.W. Cheng H.L. Brush J.W. Li G. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 4518-4523Crossref PubMed Scopus (91) Google Scholar) associated with an absence of NHEJ activity in vitro (17Buck D. Kim L. de Chasseval R. Barraud A. Fondaneche M.C. Sanal O. Plebani A. Stephan J.L. Hufnagel M. le Deist F. Fischer A. Durandy A. de Villartay J.P. Revy P. Cell. 2006; 124: 287-299Abstract Full Text Full Text PDF PubMed Scopus (579) Google Scholar, 23Dai Y. Kim B. Hanakahi L.A. Manolis K. Riballo E. Stumm M. Harville T.O. West S.C. Oettinger M.A. Jeggo P.A. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 2462-2467Crossref PubMed Scopus (154) Google Scholar). Cernunnos-defective embryonic stem cells show an impaired ability to form both V(D) J coding and signal joins in transient recombination assays (22Zha S. Kim F.W. Cheng H.L. Brush J.W. Li G. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 4518-4523Crossref PubMed Scopus (91) Google Scholar). Cernunnos-XLF is therefore likely involved in most NHEJ reactions and not just those that require end processing in contrast to DNA-PKcs and Artemis. In addition, like XRCC4 and DNA ligase IV, Cernunnos-XLF may be implicated in the development of the central nervous system (24Cantagrel V. Kim A.M. Lisgo S. Missirian C. Borges A. Philip N. Fernandez C. Cardoso C. Figarella-Branger D. Moncla A. Lindsay S. Dobyns W.B. Villard L. Hum. Mutat. 2006; 28: 356-364Crossref Scopus (29) Google Scholar). Its participation in the XRCC4/ligase IV complex, its structural resemblance with XRCC4 (18Ahnesorg P. Kim P. Jackson S.P. Cell. 2006; 124: 301-313Abstract Full Text Full Text PDF PubMed Scopus (582) Google Scholar, 19Callebaut I. Kim L. Fischer A. Mornon J.P. Revy P. de Villartay J.P. J. Biol. Chem. 2006; 281: 13857-13860Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar), and its specific stimulation of ligation in ligase IV-dependent assays (20Hentges P. Kim P. Pitcher R.S. Bruce C.K. Kysela B. Green A.J. Bianchi J. Wilson T.E. Jackson S.P. Doherty A.J. J. Biol. Chem. 2006; 281: 37517-37526Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 21Lu H. Kim U. Schwarz K. Lieber M.R. J. Biol. Chem. 2007; 282: 11155-11162Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar) suggest that this protein might function by activating or enhancing the basic NHEJ ligation reaction (25Revy P. Kim L. de Villartay J.P. Curr. Opin. Allergy Clin. Immunol. 2006; 6: 416-420Crossref PubMed Scopus (23) Google Scholar), but its precise function is still unknown.To get insight into the relationship between Cernunnos-XLF and the other members of the NHEJ process, we have analyzed the interactions of Cernunnos-XLF and NHEJ proteins in cells after treatment with double strand-breaking agents. In particular, we have used a detergent-based cellular fractionation protocol that allows assessment in situ of the DSB-induced recruitment of NHEJ repair proteins after cell treatment with IR or radiomimetic molecules (26Drouet J. Kim C. Lefrancois J. Concannon P. Salles B. Calsou P. J. Biol. Chem. 2005; 280: 7060-7069Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar).MATERIALS AND METHODSChemicals—Calicheamicin γ1 (Cali), a generous gift from P. R. Hamann (Wyeth Research, Pearl River, NY), was dissolved at 4 mm in ethanol and stored at–70 °C. Wortmannin (Sigma) and NU7026 (Calbiochem) were dissolved in Me2SO (10 mm stock solution) and stored at–20 °C. Small aliquots of stock solutions chemicals were used once.Antibodies—Polyclonal rabbit antibody anti-XLF raised against the region between amino acids 250 and 299 was from Bethyl Laboratories. Anti-Ku70 (N3H10), anti-Ku80 (clone 111), anti-p460 (DNA-PKcs, clone 18.2), and anti-β-actin (clone ACTN05) monoclonal antibodies were from Neomarkers. Monoclonal antibody antiphosphorylated H2AX (JBW301) was from Upstate Cell Signaling Solutions. Rabbit serum anti-XRCC4 was raised against full-length recombinant protein produced in baculovirus, and IgG was affinity-purified. Polyclonal rabbit antibody anti-ligase IV, anti-pS2056, and monoclonal antibody anti-γ-tubulin were from Serotec or gifts from Dr. D. J. Chen (University of Texas, Dallas) and Dr. M. Defais (Institut de Pharmacologie et de Biologie Structurale, Toulouse, France), respectively. Peroxidase-conjugated goat anti-mouse or anti-rabbit secondary antibodies were from Jackson Immuno-Research Laboratories.Cell Culture and Cloning—All culture media were from Invitrogen and were supplemented with 10% fetal calf serum unless indicated, 2 mm glutamine, 125 units/ml penicillin, and 125 μg/ml streptomycin. All cells were grown in a humidified atmosphere at 37 °C with 5% CO2. MRC5-SV2 was from the European Collection of Cell Cultures (ECACC, Salisbury, Wiltshire, UK) and was grown in Dulbecco’s modified Eagle’s medium. BuS cells are SV40T-transformed, telomerase-immortalized radiosensitive fibroblasts from the Cernunnos-deficient P2 patient as published (17Buck D. Kim L. de Chasseval R. Barraud A. Fondaneche M.C. Sanal O. Plebani A. Stephan J.L. Hufnagel M. le Deist F. Fischer A. Durandy A. de Villartay J.P. Revy P. Cell. 2006; 124: 287-299Abstract Full Text Full Text PDF PubMed Scopus (579) Google Scholar). BuC cells were obtained after transduction of BuS with the pMND-Cernunnos-Myc-ires-GFP retroviral vector expressing a C-terminal Myc-His-tagged Cernunnos protein. 4L. Malivert and P. Revy, unpublished results. Both cell lines were grown in RPMI 1640 medium. DNA-PKcs-deficient and -complemented cell lines (Fus9-alias M059J and Fus1, respectively (27Hoppe B.S. Kim R.B. Kirchgessner C.U. Radiat. Res. 2000; 153: 125-130Crossref PubMed Scopus (61) Google Scholar), a gift from Dr. C. Kirchgessner, Stanford University School of Medicine, CA) were maintained in Dulbecco’s modified Eagle’s medium-F12 1/1. The LIG4-defective N114P2 cells and the parental cell line Nalm-6 (gifts from Dr. M. R. Lieber, University of Southern California, Los Angeles) were isolated as described previously (15Grawunder U. Kim D. Fugmann S. Schwarz K. Lieber M.R. Mol. Cell. 1998; 2: 477-484Abstract Full Text Full Text PDF PubMed Scopus (282) Google Scholar) and cultured in RPMI 1640 medium. Small interfering RNA design and cloning in pEBV-based small interfering RNA vectors carrying a hygromycin B resistance cassette and establishment of knockdown and control HeLa clones were as described elsewhere (28Despras E. Kim P. Salles B. Calsou P. Kuhfittig-Kulle S. Angulo J.F. Biard D.S. Cancer Res. 2007; 67: 2526-2534Crossref PubMed Scopus (51) Google Scholar, 29Biard D.S. Nucleic Acids Res. 2007; 35: 3535-3550Crossref PubMed Scopus (59) Google Scholar). HeLa clones were grown in Dulbecco’s modified Eagle’s medium in the presence of 125 μg/ml hygromycin B (Invitrogen). The same procedure as for HeLa was followed to establish an MRC5-shL4 clone expressing a short hairpin RNA (shRNA) silencing LIG4 and a BuC-shX4 clone expressing a shRNA silencing XRCC4. The RNA-interfering sequences for LIG4 (NM_002312) and for XRCC4 (NM_022550) were nucleotides 1939 to 1957 and nucleotides 674 to 692, respectively.DNA-damaging Treatments—For drug exposure, exponentially growing cells were either mock-treated or treated with freshly diluted calicheamicin at the specified concentrations in medium at 37 °C in culture dishes and then harvested at the indicated time points. For cell treatment with IR, irradiation was carried out in a Faxitron RX-650 irradiator (Faxitron X-ray Corp., Buffalo Grove, IL) at a dose rate of 5.72 grays/min. For UV irradiation, cells were washed with phosphate-buffered saline (PBS) and then exposed to UVC irradiation (254 nm) from a germicidal lamp (Bioblock Scientific). Immediately after irradiation, unsupplemented medium was added, and cells were postincubated as above.Biochemical Fractionation and Immunoblotting—Treated or mock-treated cells in culture dishes were washed twice with ice-cold PBS, collected by scraping, and centrifuged. Cell fractionation was carried out by two consecutive extractions. Pellets of about 1 × 106 cells were first resuspended for 10 min on ice in 200 μl of extraction buffer (50 mm Hepes, pH 7.5, 150 mm NaCl, 1 mm EDTA) containing 0.1% Triton X-100, supplemented with protease inhibitor mixture tablets (Complete Mini™, Roche Diagnostics) and phosphatase inhibitors (10 mm NaF, 10 mm β-glycerophosphate, 1 mm sodium orthovanadate, and 1 mm cantharidin, all from Sigma). Following centrifugation at 14,000 × g for 3 min, the supernatant was collected (fraction S1), and the pellet was washed with extraction buffer without Triton. The pellet was further incubated in 100 μlof extraction buffer without Triton but supplemented with 200 μg/ml Rnase A (Sigma) for 30 min at 25 °C under agitation. Following centrifugation at 14,000 × g for 3 min, the pellet was washed with extraction buffer without Triton (fraction P2). Insoluble P2 fraction was resuspended in PBS buffer supplemented with 1% SDS, heated 10 min at 100 °C, and sonicated for 10 s (Vibracel, Bioblock Scientific). Whole cell extracts (WCEs) of treated or mock-treated cells were obtained by direct lysis in PBS buffer supplemented with 1% SDS and treatment as above. When necessary, the treated or mock-treated cell pellets were resuspended in 1× lambda phosphatase buffer (New England Biolabs) with 1% Triton X-100 in the presence of 2 mm magnesium chloride and protease inhibitors as above, sonicated on ice, and incubated for 1 h at 37°C with 400 units of lambda phosphatase (New England Biolabs). Concentrated loading sample buffer was added for 1× final concentration in all fractions, and the samples were boiled for 5 min. Equal aliquots of each fraction derived from equivalent cell numbers were separated on SDS-polyacrylamide gels (10% for standard separation or 15% for γ-H2AX isolation) and blotted onto polyvinylidene difluoride membranes (Immobilon-P, Millipore). Membranes were blocked for 1 h in 5% dry milk in PBS containing 0.1% Tween 20 (PBS-T) and incubated for 1 h with primary antibody diluted in PBS containing 0.02% Tween 20 and 1% bovine serum albumin (fraction V, Sigma). After three washes with PBS-T, membranes were incubated for 1 h with secondary antibodies in PBS containing 0.02% Tween 20 and 5% dry milk. Immunoblots were visualized by enhanced chemiluminescence (Immunofax A, Yelen). When necessary, successive immunoblotting was performed on the same membranes after stripping (Restore Western blot stripping buffer, Pierce). For data presentation, films were scanned and processed with Adobe PhotoShop 3.0 software.Coimmunoprecipitation Assay—Cell extracts were obtained as follows. Cells were washed with cold PBS, spun at 4 °C, 300 × g for 5 min, resuspended in hypotonic buffer HB (10 mm Hepes pH 7.5, 25 mm KCl, 10 mm NaCl, 1 mm MgCl2, 0.1 mm EDTA) supplemented with protease inhibitors (Complete Mini™, Roche Diagnostics) and phosphatase inhibitors (10 mm NaF, 10 mm β-glycerophosphate, 1 mm sodium orthovanadate, and 1 mm cantharidin, all from Sigma), and then lysed by freezing in liquid nitrogen and thawing at 37 °C three times. Lysates were then supplemented with NaCl to final 350 mm and cleared by spinning at 4 °C, 15,000 × g for 30 min. The soluble proteins were diluted with hypotonic buffer to final 120 mm NaCl, and protein concentration was measured. 100 μg of proteins were mixed in the reaction volume completed to 100 μl with immunoprecipitation buffer (10 mm Hepes, pH 7.5, 25 mm KCl, 120 mm NaCl, 1 mm MgCl2, 0.1 mm EDTA) with proteinase inhibitors and phosphatase inhibitors as above. When necessary, ethidium bromide was added at 100 μg/ml final concentration. For anti-Cernunnos-XLF immunoprecipitation, the mixture was mixed with 10 μl of magnetic anti-rabbit IgG Immunobeads suspension coated with the anti-XLF primary antibody according to the manufacturer’s protocol (Dynal), and the beads were mixed gently on a wheel for 3 h at 4°C. The beads were pulled down over a magnet, the supernatant extract was removed, the beads were washed three times with 1 ml of ice-cold PBS-T, and proteins in the immunoprecipitates were eluted by boiling in SDS sample buffer. Samples were thereafter incubated for 30 min at room temperature in the presence of iodoacetamide (100 mm) and then separated in a 10% acrylamide Tris-glycine-SDS gel. For immunodetection after transfer on polyvinylidene difluoride membranes, rabbit TrueBlot horseradish peroxidase-conjugated anti-rabbit secondary antibodies (eBioscience) were used as secondary antibodies to reduce the interfering signal of the immunoglobulins used for the immunoprecipitation.RESULTSCernunnos-XLF Is Phosphorylated by DNA-PKcs during the Cellular Response to DSBs—Cernunnos-XLF has been identified recently as a new member of the NHEJ apparatus. Because other members such as XRCC4 (26Drouet J. Kim C. Lefrancois J. Concannon P. Salles B. Calsou P. J. Biol. Chem. 2005; 280: 7060-7069Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar), Artemis (11Drouet J. Kim P. Delteil C. de Villartay J.P. Salles B. Calsou P. J. Biol. Chem. 2006; 281: 27784-27793Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar), and DNA-PKcs itself (30Chan D.W. Kim B.P. Prithivirajsingh S. Kurimasa A. Story M.D. Qin J. Chen D.J. Genes Dev. 2002; 16: 2333-2338Crossref PubMed Scopus (386) Google Scholar, 31Ding Q. Kim 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 (261) Google Scholar) are phosphorylated in the nucleus after generation of DSBs, we first addressed the possibility that Cernunnos-XLF could also be a substrate of DNA-PKcs.Cernunnos-XLF was easily detected by Western blot on WCEs from the human MRC5-SV2 fibroblasts, as compared with extracts from XLF-deficient human BuS cells carrying a truncating mutation at amino acid 177 in which it was undetectable (Fig. 1A). For production of DSBs, MRC5-SV2 cells were treated with IR or with Cali, which yields a 1:3 ratio of DNA double strand breaks to single strand breaks in vivo compared with a 1:20 ratio for IR (32Elmroth K. Kim J. Martensson S. Ismail I.H. Hammarsten O. DNA Repair (Amst.). 2003; 2: 363-374Crossref PubMed Scopus (68) Google Scholar). As opposed to non- or UV-treated cells, WCE from IR- and Cali-treated cells contained γ-H2AX, the phosphorylated form on Ser139 of the histone H2AX variant, which is admitted to be a quantitative nuclear marker of DSBs (33Rothkamm K. Kim I. Thompson L.H. Lobrich M. Mol. Cell. Biol. 2003; 23: 5706-5715Crossref PubMed Scopus (953) Google Scholar) (Fig. 1B). Under both of these latter conditions, XRCC4 exhibited a slower migrating form that was sensitive to lambda phosphatase (Fig. 1C), indicating that it corresponded to phosphorylation as reported previously (26Drouet J. Kim C. Lefrancois J. Concannon P. Salles B. Calsou P. J. Biol. Chem. 2005; 280: 7060-7069Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). Similarly, a slower migrating form of Cernunnos-XLF sensitive to lambda phosphatase was detected which was faint after IR but predominant after Cali (Fig. 1, B and C). This indicates that, like XRCC4, Cernunnos-XLF is phosphorylated in the cell in the presence of DSBs.We sought to determine whether DNA-PKcs activity was necessary for Cernunnos-XLF phosphorylation after DSBs. We first used the selective DNA-PKcs inhibitor NU7026 that has been shown to exhibit a strong DNA-PKcs-dependent radiosensitization effect on cells (34Veuger S.J. Kim N.J. Richardson C.J. Smith G.C. Durkacz B.W. Cancer Res. 2003; 63: 6008-6015PubMed Google Scholar). MRC5-SV2 cells were pretreated or not with NU7026 before treatment with Cali. As shown in Fig. 2A, the DNA-PKcs inhibitor strongly reduced both XRCC4 and Cernunnos-XLF phosphorylation. This suggests that the phosphorylation observed under these conditions mostly relies on the NU7026-sensitive DNA-PKcs activity. Then we treated with Cali the M059J glioblastoma cells that do not express DNA-PKcs (DNA-PKcs-deficient cells, Fus9) and M059J-complemented cells that contain an extra copy of the human gene coding for DNA-PKcs (DNA-PKcs-complemented cells, Fus1) (27Hoppe B.S. Kim R.B. Kirchgessner C.U. Radiat. Res. 2000; 153: 125-130Crossref PubMed Scopus (61) Google Scholar). As shown in Fig. 2B, although both XRCC4 and Cernunnos-XLF were phosphorylated upon Cali treatment of the DNA-PKcs proficient Fus1 cells, no shift was observed for either protein after treatment of the DNA-PKcs-deficient Fus9 cells. These data clearly implicate DNA-PKcs in the phosphorylation of Cernunnos-XLF upon generation of DSBs in DNA of human cells.FIGURE 2Effect of a defect in DNA-PK activity on the DSBs-induced phosphorylation of Cernunnos-XLF. A, MRC5-SV2 cells were treated or not with Cali (10 nm) for 1 h in the presence or not of NU7026 (30 μm) before lysis. Whole cell extracts were denatured and separated on 10% SDS-PAGE gels followed by electrotransfer onto membranes. The membranes were blotted with the antibodies as indicated. B, DNA-PKcs deficient (Fus9) and DNA-PKcs complemented (Fus1) glioblastoma cell lines were incubated for 1 h at 37°C with 10 nm Cali, and then cells were collected and lysed in denaturating buffer. Whole cell extracts were denatured and separated on 10% SDS-PAGE gel followed by electrotransfer onto membranes and blotting with the antibodies as indicated.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Cernunnos-XLF Is Corecruited with the Major NHEJ Components but Is Not Necessary for Their Mobilization to Chromatin Damaged with DSBs—Cernunnos-XLF has been shown to be part of a larger complex with XRCC4 and DNA ligase IV (18Ahnesorg P. Kim P. Jackson S.P. Cell. 2006; 124: 301-313Abstract Full Text Full Text PDF PubMed Scopus (582) Google Scholar, 19Callebaut I. Kim L. Fischer A. Mornon J.P. Revy P. de Villartay J.P. J. Biol. Chem. 2006; 281: 13857-13860Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). Although it stimulates ligation in a ligase IV-dependent assay (20Hentges P. Kim P. Pitcher R.S. Bruce C.K. Kysela B. Green A.J. Bianchi J. Wilson T.E. Jackson S.P. Doherty A.J. J. Biol. Chem. 2006; 281: 37517-37526Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 21Lu H. Kim U. Schwarz K. Lieber M.R. J. Biol. Chem. 2007; 282: 11155-11162Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar) and it is indispensable for the repair of DSBs in the cell (17Buck D. Kim L. de Chasseval R. Barraud A. Fondaneche M.C. Sanal O. Plebani A. Stephan J.L. Hufnagel M. le Deist F. Fischer A. Durandy A. de Villartay J.P. Revy P. Cell. 2006; 124: 287-299Abstract Full Text Full Text PDF PubMed Scopus (579) Google Scholar, 18Ahnesorg P. Kim P. Jackson S.P. Cell. 2006; 124: 301-313Abstract Full Text Full Text PDF PubMed Scopus (582) Google Scholar), the precise role of Cernunnos-XLF is still unknown. We have recently examined nuclear compartmentalization of NHEJ factors and demonstrated that in response to drugs that induce DSBs, NHEJ components mobilize to a detergent-insoluble nuclear fraction corresponding to the damaged chromatin (26Drouet J. Kim C. Lefrancois J. Concannon P. Salles B. Calsou P. J. Biol. Chem. 2005; 280: 7060-7069Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). Therefore we have addressed the question of Cernunnos-XLF mobilization to damaged chromatin together with the other NHEJ factors as a result of production of DSBs in the cell.After 1 h of treatment with calicheamicin, MRC5-SV2 cells were extracted with a buffer containing Triton; thereafter, the cell pellet was treated with RNaseA in the same buffer but without detergent, and insoluble P2 fraction was collected. A parallel extraction procedure was performed on untreated cells and cells treated with Cali. Fig. 3A shows the immunoblot analysis following SDS-PAGE of cell-equivalent aliquots of P2 fraction compared with WCEs under both untreated and Cali-treated conditions. In contrast to extracts from mock-treated cells, extracts from Cali-treated cells contain γ-H2AX consistent with the high DNA double strand-breaking potency of C" @default.
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• W2119371129 title "Interplay between Cernunnos-XLF and Nonhomologous End-joining Proteins at DNA Ends in the Cell" @default.
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