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• W2137613124 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 redistribution we transiently expressed wild-type H2AX or a S140A phosphomutant in H2AX-deficient ES cells and assessed 53BP1 foci formation. As shown in Fig. 2A, expression of wild-type H2AX reconstituted 53BP1 foci formation in response to IR. In marked contrast, expression of the H2AX S140A phosphomutant was insufficient to induce 53BP1 accumulation at the sites of DNA strand breaks. We had shown earlier that phosphorylated H2AX co-immunoprecipitates 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 focus localization interacts directly with γ-H2AX, we used an in vitro pull-down assay. Six different 53BP1 GST fragments spanning the entire 53BP1 protein were incubated with immobilized C-terminal H2AX peptide that was either phosphorylated or non-phosphorylated at Ser-140. Only 53BP1 fragment 956–1354, which overlaps with the mapped focus localization region, showed strong interaction with the phosphorylated H2AX peptide (Fig. 2B). As a control, no binding was detected to the non-phosphorylated peptide bearing identical sequence (Fig. 2B). Since H2AX directs 53BP1 accumulation in response to DNA damage, we asked whether H2AX is also required for the kinetochore localization of 53BP1 in mitotic cells (19Jullien D. Vagnarelli P. Earnshaw W.C. Adachi Y. J. Cell Sci. 2002; 115: 71-79Crossref PubMed Google Scholar). As shown in Fig. 2C, 53BP1 can be readily detected at the kinetochores in H2AX-deficient mitotic cells suggesting that the kinetochore localization of 53BP1 is not mediated by phospho-H2AX. Phosphorylation of 53BP1 Is Not Required for Foci Formation—We had previously demonstrated that 53BP1 becomes hyperphosphorylated in response to IR, and three 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 map the phosphorylation sites we designed a series of GST fusion peptides containing one or two ATM binding motifs (SQ or TQ sites). ATM kinase assays using these purified GST fusion proteins as substrates, and ATM kinase immunoprecipitated from either K562 lysates (containing wild-type ATM) or ATM-deficient GM03189D lysates revealed peptides aa 1–12, aa 18–37, and aa 778–791 as putative ATM substrates (Fig. 3A). To examine whether the respective SQ sites become phosphorylated in vivo, we raised polyclonal antibodies against phosphorylated Ser-6 (anti-S6P), phosphorylated Ser-25 and Ser-29 (anti-S25P/29P), and phosphorylated Ser-784 (anti-S784P). All affinity-purified antisera recognized 53BP1 in irradiated cells but not in untreated controls when assessed by immunofluorescence analysis (data not shown). In addition, anti-53BP1 S25P/29P antibodies detected 53BP1 from irradiated ATM wild-type but not ATM-deficient cells by Western blot analyses (Fig. 3B). Pretreatment with λ-phosphatase abolished the antibody binding further validating that anti-53BP1 S25P/29P specifically recognizes the phosphorylated form of 53BP1 (Fig. 3B). To test whether phosphorylation of 53BP1 is required for the recruitment of 53BP1 to sites of DNA lesions, we generated a phosphorylation-deficient mutant (53BP1 4SA) by mutating the mapped ATM target sites (Ser-6, Ser-25/Ser-29, and Ser-784) to alanines. 53BP1–/– embryonic cells transfected with this phosphomutant showed normal 53BP1 foci formation in response to IR (Fig. 3C), indicating that ATM-dependent phosphorylation of 53BP1 is not required for recruitment to or retention of 53BP1 at DNA break sites. Phosphorylation of 53BP1 might occur at the break areas. To test this possibility, we transiently transfected 53BP1-deficient embryonic cells with the HA-tagged mutant that lacks part of the 53BP1 focus localization region (Δ1052–1305) and remains a diffuse nuclear localization upon exposure of cells to IR. Co-immunostaining with anti-HA and anti-53BP1 S25P/29P antibodies revealed that ATM-dependent 53BP1 phosphorylation does not require 53BP1 foci formation (Fig. 3D). Immunoprecipitation assays confirmed that 53BP1 Δ1052–1305 becomes readily phosphorylated at Ser-25/Ser-29 in response to IR (Fig. 3E). These findings are consistent with a recent report describing phosphorylation of 53BP1 Ser-25 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). Taken together, these results suggest that 53BP1 focus localization and ATM-dependent phosphorylation of 53BP1 are regulated independently. Upon exposure of cells to genotoxic stress, 53BP1 rapidly redistributes from a pan-nuclear 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 accumulation. Moreover, we provided evidence that phosphorylation of 53BP1 by the ATM kinase occurs independently of 53BP1 foci formation. 53BP1 had been speculated to be involved in the DNA damage response based on its C-terminal tandem BRCT domains. This motif was first detected in the BRCA1 C terminus and has been reported to bind directly to DNA breaks (20Yamane K. Katayama E. Tsuruo T. Biochem. Biophys. Res. Commun. 2000; 279: 678-684Crossref PubMed Scopus (49) Google Scholar). Surprisingly the BRCT domains of 53BP1 were found to be dispensable for 53BP1 foci formation. Instead a region upstream of the BRCT motifs proved to be essential for 53BP1 accumulation at sites of DNA strand breaks. Our data suggest that the damage-induced phosphorylation of H2AX directs 53BP1 accumulation at sites of DNA strand breaks. First, H2AX-deficient cells reconstituted with a H2AX phosphomutant failed to induce or sustain 53BP1 foci formation. Second, a 53BP1 fragment (residues 956–1354) contained within the 53BP1 focus localization region interacted strongly with phosphorylated H2AX in vitro. Third, 53BP1 co-immunoprecipitates with H2AX in a DNA damage-dependent manner (3Rappold I. Iwabuchi K. Date T. Chen J. J. Cell Biol. 2001; 153: 613-620Crossref PubMed Scopus (414) Google Scholar). Thus, it is likely that the DNA damage-induced phosphorylation of H2AX at Ser-140 increases the interaction between H2AX and 53BP1 and leads to the accumulation of 53BP1 at the sites of DNA breaks. Interestingly the focus localization region we mapped includes a region required for 53BP1 kinetochore localization in mitotic cells (residues 1220–1601) (19Jullien D. Vagnarelli P. Earnshaw W.C. Adachi Y. J. Cell Sci. 2002; 115: 71-79Crossref PubMed Google Scholar). Very recently, Morales and colleagues (21Morales J.C. Xia Z. Lu T. Aldrich M.B. Wang B. Rosales C. Kellems R.E. Hittelman W.N. Elledge S.J. Carpenter P.B. J. Biol. Chem. 2003; 278: 14971-14977Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar) showed that the kinetochore localization region is also essential for 53BP1 foci formation in response to DNA damage suggesting that both events might be regulated in a similar fashion. However, the kinetochore localization of 53BP1 is unlikely to involve DNA lesions (19Jullien D. Vagnarelli P. Earnshaw W.C. Adachi Y. J. Cell Sci. 2002; 115: 71-79Crossref PubMed Google Scholar). We have shown that 53BP1 kinetochore localization appears normal in H2AX-deficient cells, suggesting that kinetochore localization of 53BP1 is not mediated by phospho-H2AX. Further fine mapping studies will be necessary to clarify whether the same 53BP1 region initiates the recruitment of 53BP1 to DNA strand breaks or the kinetochore, respectively. Phosphorylation by the ATM kinase plays a key role in the activation of various proteins involved in the DNA damage response (for example, see Ref. 22Abraham R.T. Genes Dev. 2001; 15: 2177-2196Crossref PubMed Scopus (1682) Google Scholar). A recent study by Bakkenist and Kastan (23Bakkenist C.J. Kastan M.B. Nature. 2003; 421: 499-506Crossref PubMed Scopus (2725) Google Scholar) revealed that ATM forms an inactive oligomer in unirradiated cells. Upon radiation, ATM is rapidly autophosphorylated and dissociates from the complex thereby providing other substrates access to its kinase domain. Interestingly autophoshorylation and activation of ATM seem to occur at some distance to DNA break sites, and ATM then migrates in the nucleus to phosphorylate various substrates either at the break sites or elsewhere in the nucleus (23Bakkenist C.J. Kastan M.B. Nature. 2003; 421: 499-506Crossref PubMed Scopus (2725) Google Scholar). This model is consistent with our finding that ATM-dependent phosphorylation of 53BP1 is not restricted to sites of DNA strand breaks and can occur within the entire nucleus. However, phosphorylation of 53BP1 alone is unlikely to trigger 53BP1 activation since deletion of the ATM target sites does not affect 53BP1 relocalization. Moreover the relocalization of 53BP1 appears to be required for efficient phosphorylation of ATM substrates at the sites of DNA breaks (data not shown). We therefore speculate that the rapid recruitment of 53BP1 to DNA break sites and the retention of 53BP1 at the sites of DNA breaks by binding to phospho-H2AX is one of the key steps in the activation of 53BP1 following DNA damage. We thank Dr. Craig Bassing for the H2AXFlox/Flox and H2AXΔ/Δ ES cells. We also thank Drs. Larry Karnitz and Scott Kaufmann and members of the Chen and Karnitz laboratories for helpful discussions. We are grateful to the Mayo Protein Core facility for the synthesis of peptides and to the Mayo Gene Targeted Mouse Core facility for help with the generation of 53BP1-deficient mice and 53BP1-deficient ES cells." @default.
• W2137613124 created "2016-06-24" @default.
• W2137613124 creator A5029892501 @default.
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• W2137613124 date "2003-05-01" @default.
• W2137613124 modified "2023-09-30" @default.
• W2137613124 title "Accumulation of Checkpoint Protein 53BP1 at DNA Breaks Involves Its Binding to Phosphorylated Histone H2AX" @default.
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• W2137613124 doi "https://doi.org/10.1074/jbc.c300117200" @default.
• W2137613124 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/12697768" @default.
• W2137613124 hasPublicationYear "2003" @default.
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