Accumulation of Checkpoint Protein 53BP1 at DNA Breaks Involves Its Binding to Phosphorylated Histone H2AX

Abstract

53BP1 participates in the cellular response to DNA damage. Like many proteins involved in the DNA damage response, 53BP1 becomes hyperphosphorylated after radiation and colocalizes with phosphorylated H2AX in megabase regions surrounding the sites of DNA strand breaks. However, it is not yet clear whether the phosphorylation status of 53BP1 determines its localization or vice versa. In this study we mapped a region upstream of the 53BP1 C terminus that is required and sufficient for the recruitment of 53BP1 to these DNA break areas. In vitro assays revealed that this region binds to phosphorylated but not unphosphorylated H2AX. Moreover, using H2AX-deficient cells reconstituted with wild-type or a phosphorylation-deficient mutant of H2AX, we have shown that phosphorylation of H2AX at serine 140 is critical for efficient 53BP1 foci formation, implying that a direct interaction between 53BP1 and phosphorylated H2AX is required for the accumulation of 53BP1 at DNA break sites. On the other hand, radiation-induced phosphorylation of the 53BP1 N terminus by the ATM (ataxia-telangiectasia mutated) kinase is not essential for 53BP1 foci formation and takes place independently of 53BP1 redistribution. Thus, these two damage-induced events, hyperphosphorylation and relocalization of 53BP1, occur independently in the cell. 53BP1 participates in the cellular response to DNA damage. Like many proteins involved in the DNA damage response, 53BP1 becomes hyperphosphorylated after radiation and colocalizes with phosphorylated H2AX in megabase regions surrounding the sites of DNA strand breaks. However, it is not yet clear whether the phosphorylation status of 53BP1 determines its localization or vice versa. In this study we mapped a region upstream of the 53BP1 C terminus that is required and sufficient for the recruitment of 53BP1 to these DNA break areas. In vitro assays revealed that this region binds to phosphorylated but not unphosphorylated H2AX. Moreover, using H2AX-deficient cells reconstituted with wild-type or a phosphorylation-deficient mutant of H2AX, we have shown that phosphorylation of H2AX at serine 140 is critical for efficient 53BP1 foci formation, implying that a direct interaction between 53BP1 and phosphorylated H2AX is required for the accumulation of 53BP1 at DNA break sites. On the other hand, radiation-induced phosphorylation of the 53BP1 N terminus by the ATM (ataxia-telangiectasia mutated) kinase is not essential for 53BP1 foci formation and takes place independently of 53BP1 redistribution. Thus, these two damage-induced events, hyperphosphorylation and relocalization of 53BP1, occur independently in the cell. The DNA of eukaryotic cells is constantly exposed to endogenous and exogenous DNA-damaging agents. To prevent the accumulation of genomic damage and avert cellular dysfunction, cells have evolved complex response mechanisms. 53BP1 was initially identified as a protein that binds to the central DNA binding domain of p53 and enhances p53-mediated transcriptional activation (1Iwabuchi K. Bartel P.L. Li B. Marraccino R. Fields S. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6098-6102Crossref PubMed Scopus (363) Google Scholar, 2Iwabuchi K. Li B. Massa H.F. Trask B.J. Date T. Fields S. J. Biol. Chem. 1998; 273: 26061-26068Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar). In response to genotoxic stress, 53BP1 rapidly redistributes from a diffuse nuclear localization into distinct nuclear foci suggesting that 53BP1 is involved in the DNA damage response (3Rappold I. Iwabuchi K. Date T. Chen J. J. Cell Biol. 2001; 153: 613-620Crossref PubMed Scopus (414) Google Scholar, 4Schultz L.B. Chehab N.H. Malikzay A. Halazonetis T.D. J. Cell Biol. 2000; 151: 1381-1390Crossref PubMed Scopus (725) Google Scholar, 5Xia Z. Morales J.C. Dunphy W.G. Carpenter P.B. J. Biol. Chem. 2000; 276: 2708-2718Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 6Anderson L. Henderson C. Adachi Y. Mol. Cell. Biol. 2001; 21: 1719-1729Crossref PubMed Scopus (297) Google Scholar). Moreover the C terminus of 53BP1 contains two BRCT domains, a motif found in a number of proteins implicated in various aspects of cell cycle control, recombination, and DNA repair (7Bork P. Hofmann K. Bucher P. Neuwald A.F. Altschul S.F. Koonin E.V. FASEB J. 1997; 11: 68-76Crossref PubMed Scopus (661) Google Scholar, 8Callebaut I. Mornon J.P. FEBS Lett. 1997; 400: 25-30Crossref PubMed Scopus (486) Google Scholar). Subsequent studies have shown that 53BP1 becomes hyperphosphorylated in response to ionizing radiation (IR) 1The abbreviations used are: IR, ionizing radiation; γ-H2AX, phosphorylated histone H2AX; ATM, ataxia-telangiectasia mutated; NLS, nuclear localization sequence; HA, hemagglutinin; ES, embryonic stem; KLH, keyhole limpet hemocyanin; Gy, gray(s); GST, glutathione S-transferase; aa, amino acid(s); IF, immunofluorescence; BRCT, BRCA1 C terminus. and colocalizes with phosphorylated histone H2AX (γ-H2AX) at the sites of DNA lesions (3Rappold I. Iwabuchi K. Date T. Chen J. J. Cell Biol. 2001; 153: 613-620Crossref PubMed Scopus (414) Google Scholar, 4Schultz L.B. Chehab N.H. Malikzay A. Halazonetis T.D. J. Cell Biol. 2000; 151: 1381-1390Crossref PubMed Scopus (725) Google Scholar). Other proteins known to be involved in the DNA damage signaling pathway (i.e. BRCA1, Rad51, NBS1, and TopoBP1) were also found to colocalize with 53BP1 in these inducible foci (3Rappold I. Iwabuchi K. Date T. Chen J. J. Cell Biol. 2001; 153: 613-620Crossref PubMed Scopus (414) Google Scholar, 4Schultz L.B. Chehab N.H. Malikzay A. Halazonetis T.D. J. Cell Biol. 2000; 151: 1381-1390Crossref PubMed Scopus (725) Google Scholar, 6Anderson L. Henderson C. Adachi Y. Mol. Cell. Biol. 2001; 21: 1719-1729Crossref PubMed Scopus (297) Google Scholar, 9Yamane K. Wu X. Chen J. Mol. Cell. Biol. 2002; 22: 555-566Crossref PubMed Scopus (154) Google Scholar). Direct evidence for an important role of 53BP1 in the DNA damage response came recently from studies using 53BP1-deficient cells. Human cell lines treated with specific small interfering RNA to silence 53BP1 expression exhibited a defect in the intra-S phase checkpoint and, at low IR doses, a partial defect in the G2-M checkpoint (10DiTullio R.A. Mochan T.A. Venere M. Bartkova J. Sehested M. Bartek J. Halazonetis T.D. Nat. Cell Biol. 2002; 4: 998-1002Crossref PubMed Scopus (357) Google Scholar, 11Wang B. Matsuoka S. Carpenter P.B. Elledge S.J. Science. 2002; 298: 1435-1438Crossref PubMed Scopus (484) Google Scholar, 12Fernandez-Capetillo O. Chen H.T. Celeste A. Ward I. Romanienko P.J. Morales J.C. Naka K. Xia Z. Camerini-Otero R.D. Motoyama N. Carpenter P.B. Bonner W.M. Chen J. Nussenzweig A. Nat. Cell Biol. 2002; 4: 993-997Crossref PubMed Scopus (580) Google Scholar). Moreover 53BP1-deficient mice are hypersensitive to ionizing radiation and show an increased incidence of developing thymic lymphomas (13Ward I.M. Minn K. Van Deursen J. Chen J. Mol. Cell. Biol. 2003; 23: 2556-2563Crossref PubMed Scopus (331) Google Scholar). Several lines of evidence suggest that 53BP1 is a substrate of ATM, the kinase mutated in the human disease ataxia-telangiectasia, and is involved in the phosphorylation of various ATM substrates (3Rappold I. Iwabuchi K. Date T. Chen J. J. Cell Biol. 2001; 153: 613-620Crossref PubMed Scopus (414) Google Scholar, 6Anderson L. Henderson C. Adachi Y. Mol. Cell. Biol. 2001; 21: 1719-1729Crossref PubMed Scopus (297) Google Scholar, 10DiTullio R.A. Mochan T.A. Venere M. Bartkova J. Sehested M. Bartek J. Halazonetis T.D. Nat. Cell Biol. 2002; 4: 998-1002Crossref PubMed Scopus (357) Google Scholar). Despite this recent progress made toward 53BP1 function little is known about the initial activation of 53BP1. Recruitment of 53BP1 to γ-H2AX foci seems to be a crucial step. H2AX-deficient cells lack normal 53BP1 foci formation and, like 53BP1-deficent cells, manifest a G2-M checkpoint defect after exposure to low doses of ionizing radiation (12Fernandez-Capetillo O. Chen H.T. Celeste A. Ward I. Romanienko P.J. Morales J.C. Naka K. Xia Z. Camerini-Otero R.D. Motoyama N. Carpenter P.B. Bonner W.M. Chen J. Nussenzweig A. Nat. Cell Biol. 2002; 4: 993-997Crossref PubMed Scopus (580) Google Scholar). Moreover, H2AX–/– mice show a radiation sensitivity similar to 53BP1–/– mice (14Celeste A. Petersen S. Romanienko P.J. Fernandez-Capetillo O. Chen H.T. Sedelnikova O.A. Reina-San-Martin B. Coppola V. Meffre E. Difilippantonio M.J. Redon C. Pilch D.R. Olaru A. Eckhaus M. Camerini-Otero R.D. Tessarollo L. Livak F. Manova K. Bonner W.M. Nussenzweig M.C. Nussenzweig A. Science. 2002; 296: 922-927Crossref PubMed Scopus (1147) Google Scholar). In this study we mapped the region required for 53BP1 foci formation in response to DNA damage. We show that a region upstream of the BRCT motifs is sufficient for 53BP1 foci formation and that this region interacts directly with phosphorylated H2AX. Using H2AX-deficient cells retransfected with either wild-type H2AX or an H2AX phosphomutant we confirm that phosphorylation of H2AX at Ser-140 is required for 53BP1 accumulation at DNA break areas. In contrast, radiation-induced phosphorylation of 53BP1 by ATM is not essential for the recruitment of 53BP1 to foci and occurs independently. Plasmid Constructs and Transfection—53BP1 deletion mutants were generated by inserting stop codons and/or restriction sites at various positions into pCMH6K 53BP1 (2Iwabuchi K. Li B. Massa H.F. Trask B.J. Date T. Fields S. J. Biol. Chem. 1998; 273: 26061-26068Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar) using the QuikChange site-directed mutagenesis kit (Stratagene). A 3×NLS (nuclear localization sequence) was inserted into the NheI site upstream of the N-terminal hemagglutinin (HA) and His6 tags. U2OS cells were transfected using FuGENE 6 (Roche Applied Science) according to the manufacturer's instructions. H2AX was PCR-amplified from human genomic DNA and inserted between the HA tag and an internal ribosomal entry site fused to the puromycin gene of a modified pcDNA3 vector. The H2AX phosphomutant was generated by replacing Ser-140 of H2AX with an alanine using the QuikChange mutagenesis kit (Stratagene). H2AX-deficient embryonic stem (ES) cells (provided by C. Bassing, Ref. 15Bassing C.H. Chua K.F. Sekiguchi J. Suh H. Whitlow S.R. Fleming J.C. Monroe B.C. Ciccone D.N. Yan C. Vlasakova K. Livingston D.M. Ferguson D.O. Scully R. Alt F.W. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 8173-8178Crossref PubMed Scopus (463) Google Scholar) were transfected by electroporation. Antibodies—Anti-S6P, anti-S25P/29P, and anti-S784P specific antibodies were generated by coupling synthetic 53BP1 peptides (S6P, CDPTG(P)SQLD; S25P/29P, CIED(P)SQPE(P)SQVLEDD; S784P, CSD(P)SQSWEDI where (P)S represents phosphoserine) to KLH using Imject maleimide-activated mcKLH (Pierce) prior to immunizing rabbits (Cocalico Biological). The antibodies were affinity-purified on agarose columns coupled with the non-phosphorylated or phosphorylated peptide (SulfoLink Coupling Gel, Pierce). Anti-53BP1 and anti-γ-H2AX antibodies were generated as described previously (3Rappold I. Iwabuchi K. Date T. Chen J. J. Cell Biol. 2001; 153: 613-620Crossref PubMed Scopus (414) Google Scholar). Monoclonal antibody HA11 specific for HA was purchased from BabCO Berkeley Antibody Co. Anti-ATM antibody Ab3 was purchased from Oncogene Research Products. Immunofluorescence Staining, Immunoblots, and Immunoprecipitation—Cells grown on coverslips were fixed with 3% paraformaldehyde 1 h after exposure to 0 or 1 Gy of IR. After permeabilization with 0.5% Triton X-100, cells were blocked with 5% goat serum and incubated successively with the primary and secondary antibodies, each for 25 min at 37 °C. In case of DNase or RNase treatment, cells were irradiated, permeabilized with 0.5% Triton X-100 for 3 min, and incubated with either DNase I (10 units/ml) or RNase A (50 μg/ml) in phosphate-buffered saline plus calcium and magnesium for 30 min at 37 °C prior to fixation with 3% paraformaldehyde. Immunoprecipitation and immunoblot assays were done as described previously (3Rappold I. Iwabuchi K. Date T. Chen J. J. Cell Biol. 2001; 153: 613-620Crossref PubMed Scopus (414) Google Scholar). ATM Kinase Assays—ATM was precipitated from K562 cells, and aliquots of the ATM-protein A-Sepharose immunocomplex were resuspended in 25 μl of kinase buffer (10 mm Hepes (pH 7.4), 50 mm NaCl, 10 mm MgCl2, 10 mm MnCl2, 1 mm dithiothreitol, 10 nm ATP). ATM kinase reactions were carried out at 30 °C for 20 min with 10 μCi of [γ-32P]ATP and 1 μg of 53BP1 GST fusion proteins. GST Pull-down Assays—GST pull-down experiments were performed by incubating 3 μg of various GST-labeled 53BP1 fragments with C-terminal H2AX peptide that was either phosphorylated or unphosphorylated at Ser-140 (CKATQA(P)SQEY) and had been immobilized on SulfoLink Coupling Gel (Pierce). Bound GST proteins were isolated by incubating the mixture for 1 h at 4 °C in 200 μl of NETN buffer (150 mm NaCl, 1 mm EDTA, 20 mm Tris (pH 8), 0.5% Nonidet P-40), washing five times with NETN, eluting the proteins with 2× Laemmli buffer, separating them by SDS-PAGE, and immunoblotting with horseradish peroxidase-conjugated anti-GST (B-14, Santa Cruz Biotechnology). Generation of 53BP1-deficient Embryonic Cells—A 53BP1-deficient embryonic cell line was derived from 53BP1–/– blastocysts using a standard procedure. The generation of 53BP1-deficient mice is described in Ref. 13Ward I.M. Minn K. Van Deursen J. Chen J. Mol. Cell. Biol. 2003; 23: 2556-2563Crossref PubMed Scopus (331) Google Scholar. A Region Upstream of the Tandem BRCT Motif Is Required and Sufficient for 53BP1 Foci Formation—53BP1 is a large 1972-aa nuclear protein with a C-terminal tandem BRCT motif. Upstream of the BRCT repeats resides a bipartite nuclear localization signal (predictNLS, Ref. 16Cokol M. Nair R. Rost B. EMBO Rep. 2000; 1: 411-415Crossref PubMed Scopus (559) Google Scholar) and a tudor domain (RPS-BLAST, Ref. 17Altschul S.F. Madden T.L. Schaffer A.A. Zhang J. Zhang Z. Miller W. Lipman D.J. Nucleic Acids Res. 1997; 25: 3389-3402Crossref PubMed Scopus (60532) Google Scholar), a motif found in several RNA-binding proteins. In response to IR, 53BP1 rapidly redistributes to distinct nuclear foci that colocalize with γ-H2AX (3Rappold I. Iwabuchi K. Date T. Chen J. J. Cell Biol. 2001; 153: 613-620Crossref PubMed Scopus (414) Google Scholar, 4Schultz L.B. Chehab N.H. Malikzay A. Halazonetis T.D. J. Cell Biol. 2000; 151: 1381-1390Crossref PubMed Scopus (725) Google Scholar, 6Anderson L. Henderson C. Adachi Y. Mol. Cell. Biol. 2001; 21: 1719-1729Crossref PubMed Scopus (297) Google Scholar). Treatment of irradiated cells with DNase, but not RNase, completely abolished 53BP1 and γ-H2AX foci formation confirming that these foci localize to DNA (Fig. 1A). To determine the minimal region required for the recruitment of 53BP1 to damage-induced foci, we generated various HA-tagged 53BP1 deletion mutants and examined their distribution in transiently transfected U2OS cells (Fig. 1, B and C, and data not shown). A 3×NLS motif fused to the N terminus of 53BP1 ensured nuclear expression of the various constructs. Truncation of the 53BP1 N terminus (Δ1–1052) or the BRCT domains (Δ1700–1972) did not affect 53BP1 foci formation as assessed by IF 1 h after exposure to 1 Gy of IR (Fig. 1C). However, increasing C-terminal deletions (Δ1305–1972 and Δ1052–1972) or deletion of a region upstream of the tandem BRCT motifs (Δ1052–1305) abolished 53BP1 foci formation (Fig. 2, B and C, and data not shown). Moreover a 53BP1 construct expressing residues 1052–1639 including the tudor domain was found to be sufficient for 53BP1 foci formation (Fig. 1, B and C) suggesting that the domain required for foci formation is contained within this region. 53BP1 Focus Localization Region Interacts Directly with γ-H2AX—H2AX-deficient cells show greatly reduced 53BP1 foci formation implying that H2AX is involved in the recruitment of 53BP1 to radiation-induced foci (14Celeste A. Petersen S. Romanienko P.J. Fernandez-Capetillo O. Chen H.T. Sedelnikova O.A. Reina-San-Martin B. Coppola V. Meffre E. Difilippantonio M.J. Redon C. Pilch D.R. Olaru A. Eckhaus M. Camerini-Otero R.D. Tessarollo L. Livak F. Manova K. Bonner W.M. Nussenzweig M.C. Nussenzweig A. Science. 2002; 296: 922-927Crossref PubMed Scopus (1147) Google Scholar). H2AX becomes phosphorylated at a conserved C-terminal SQ site upon exposure of cells to ionizing radiation (18Rogakou E.P. Pilch D.R. Orr A.H. Ivanova V.S. Bonner W.M. J. Biol. Chem. 1998; 273: 5858-5868Abstract Full Text Full Text PDF PubMed Scopus (4236) Google Scholar). Phosphorylation of H2AX at Ser-140 is impaired in ATM-deficient cells suggesting that this site is dominantly phosphorylated by ATM (12Fernandez-Capetillo O. Chen H.T. Celeste A. Ward I. Romanienko P.J. Morales J.C. Naka K. Xia Z. Camerini-Otero R.D. Motoyama N. Carpenter P.B. Bonner W.M. Chen J. Nussenzweig A. Nat. Cell Biol. 2002; 4: 993-997Crossref PubMed Scopus (580) Google Scholar). To analyze whether phosphorylation of H2AX at Ser-140 is required for 53BP1 we transiently wild-type H2AX or a phosphomutant in H2AX-deficient cells and assessed 53BP1 foci shown in expression of wild-type H2AX reconstituted 53BP1 foci formation in response to IR. In contrast, expression of the H2AX phosphomutant was to 53BP1 accumulation at the sites of DNA strand breaks. We had shown that phosphorylated H2AX with 53BP1 upon exposure of cells to DNA damage (3Rappold I. Iwabuchi K. Date T. Chen J. J. Cell Biol. 2001; 153: 613-620Crossref PubMed Scopus (414) Google Scholar). To determine whether the region required for 53BP1 localization interacts directly with γ-H2AX, we used an in vitro pull-down 53BP1 GST fragments the 53BP1 protein were incubated with immobilized C-terminal H2AX peptide that was either phosphorylated or non-phosphorylated at 53BP1 with the mapped localization interaction with the phosphorylated H2AX peptide (Fig. a control, binding was to the non-phosphorylated peptide (Fig. H2AX 53BP1 accumulation in response to DNA we whether H2AX is also required for the localization of 53BP1 in cells P. Adachi Y. J. Cell Sci. 2002; PubMed Google Scholar). shown in 53BP1 be at the in H2AX-deficient cells suggesting that the localization of 53BP1 is not by Phosphorylation of 53BP1 Is Required for Foci had previously that 53BP1 becomes hyperphosphorylated in response to IR, and regions at the N terminus of 53BP1 were found to be phosphorylated by ATM in vitro (3Rappold I. Iwabuchi K. Date T. Chen J. J. Cell Biol. 2001; 153: 613-620Crossref PubMed Scopus (414) Google Scholar). To the phosphorylation sites we a of GST fusion peptides or two ATM binding motifs or ATM kinase assays using these GST fusion proteins as and ATM kinase from either K562 wild-type or ATM-deficient revealed peptides and as ATM substrates (Fig. To whether the SQ sites phosphorylated in we antibodies phosphorylated phosphorylated and and phosphorylated affinity-purified 53BP1 in irradiated cells but not in assessed by not shown). In antibodies 53BP1 from irradiated ATM wild-type but not ATM-deficient cells by (Fig. with abolished the antibody binding that the phosphorylated of 53BP1 (Fig. To whether phosphorylation of 53BP1 is required for the recruitment of 53BP1 to sites of DNA we generated a phosphorylation-deficient mutant by the mapped ATM sites and to 53BP1–/– embryonic cells transfected with this phosphomutant normal 53BP1 foci formation in response to IR (Fig. that phosphorylation of 53BP1 is not required for recruitment to or of 53BP1 at DNA break sites. Phosphorylation of 53BP1 occur at the break areas. To this we transiently transfected 53BP1-deficient embryonic cells with the HA-tagged mutant that of the 53BP1 localization region (Δ1052–1305) and a diffuse nuclear localization upon exposure of cells to IR. with and antibodies revealed that 53BP1 phosphorylation not 53BP1 foci formation (Fig. Immunoprecipitation assays that 53BP1 becomes phosphorylated at in response to IR (Fig. are with a recent phosphorylation of 53BP1 in H2AX-deficient cells (12Fernandez-Capetillo O. Chen H.T. Celeste A. Ward I. Romanienko P.J. Morales J.C. Naka K. Xia Z. Camerini-Otero R.D. Motoyama N. Carpenter P.B. Bonner W.M. Chen J. Nussenzweig A. Nat. Cell Biol. 2002; 4: 993-997Crossref PubMed Scopus (580) Google Scholar). these suggest that 53BP1 localization and phosphorylation of 53BP1 are independently. exposure of cells to genotoxic stress, 53BP1 rapidly redistributes from a localization to distinct nuclear foci at the sites of DNA strand breaks. In this study we have mapped the region required for 53BP1 foci formation and examined the role of H2AX in 53BP1 Moreover, we evidence that phosphorylation of 53BP1 by the ATM kinase occurs independently of 53BP1 foci 53BP1 had been to be involved in the DNA damage response on its C-terminal tandem BRCT motif was in the BRCA1 C terminus and been to directly to DNA K. E. T. Res. 2000; PubMed Scopus Google Scholar). the BRCT domains of 53BP1 were found to be for 53BP1 foci a region upstream of the BRCT motifs to be essential for 53BP1 accumulation at sites of DNA strand breaks. data suggest that the damage-induced phosphorylation of H2AX 53BP1 accumulation at sites of DNA strand breaks. H2AX-deficient cells reconstituted with a H2AX phosphomutant to or 53BP1 foci a 53BP1 contained within the 53BP1 localization region with phosphorylated H2AX in 53BP1 with H2AX in a DNA (3Rappold I. Iwabuchi K. Date T. Chen J. J. Cell Biol. 2001; 153: 613-620Crossref PubMed Scopus (414) Google Scholar). Thus, it is that the DNA damage-induced phosphorylation of H2AX at Ser-140 the interaction between H2AX and 53BP1 and to the accumulation of 53BP1 at the sites of DNA breaks. the localization region we mapped a region required for 53BP1 localization in cells P. Adachi Y. J. Cell Sci. 2002; PubMed Google Scholar). Morales and J.C. Xia Z. T. B. C. Elledge S.J. Carpenter P.B. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar) that the localization region is also essential for 53BP1 foci formation in response to DNA damage suggesting that be in a similar However, the localization of 53BP1 is to DNA lesions P. Adachi Y. J. Cell Sci. 2002; PubMed Google Scholar). We have shown that 53BP1 localization normal in H2AX-deficient cells, suggesting that localization of 53BP1 is not by studies be to whether the 53BP1 region the recruitment of 53BP1 to DNA strand or the Phosphorylation by the ATM kinase a role in the activation of various proteins involved in the DNA damage response Ref. 2001; PubMed Scopus Google Scholar). A recent study by and 2003; PubMed Scopus Google Scholar) revealed that ATM an in cells. ATM is rapidly and from the complex other substrates to its kinase and activation of ATM to occur at to DNA break and ATM in the to various substrates either at the break sites or in the 2003; PubMed Scopus Google Scholar). is with that phosphorylation of 53BP1 is not to sites of DNA strand and occur within the However, phosphorylation of 53BP1 is to 53BP1 activation deletion of the ATM sites not affect 53BP1 Moreover the relocalization of 53BP1 to be required for efficient phosphorylation of ATM substrates at the sites of DNA not shown). We that the recruitment of 53BP1 to DNA break sites and the of 53BP1 at the sites of DNA by binding to is of the in the activation of 53BP1 DNA damage. We for the and cells. We also and and of the Chen and for We are to the for the of peptides and to the for with the generation of 53BP1-deficient mice and 53BP1-deficient cells.