ATM Phosphorylates Histone H2AX in Response to DNA Double-strand Breaks

Abstract

A very early step in the response of mammalian cells to DNA double-strand breaks is the phosphorylation of histone H2AX at serine 139 at the sites of DNA damage. Although the phosphatidylinositol 3-kinases, DNA-PK (DNA-dependent proteinkinase), ATM (ataxia telangiectasiamutated), and ATR (ATM andRad3-related), have all been implicated in H2AX phosphorylation, the specific kinase involved has not yet been identified. To definitively identify the specific kinase(s) that phosphorylates H2AX in vivo, we have utilized DNA-PKcs−/− and Atm−/− cell lines and mouse embryonic fibroblasts. We find that H2AX phosphorylation and nuclear focus formation are normal in DNA-PKcs−/− cells and severely compromised in Atm−/− cells. We also find that ATM can phosphorylate H2AX in vitro and that ectopic expression of ATM in Atm−/− fibroblasts restores H2AX phosphorylation in vivo. The minimal H2AX phosphorylation in Atm−/− fibroblasts can be abolished by low concentrations of wortmannin suggesting that DNA-PK, rather than ATR, is responsible for low levels of H2AX phosphorylation in the absence of ATM. Our results clearly establish ATM as the major kinase involved in the phosphorylation of H2AX and suggest that ATM is one of the earliest kinases to be activated in the cellular response to double-strand breaks. A very early step in the response of mammalian cells to DNA double-strand breaks is the phosphorylation of histone H2AX at serine 139 at the sites of DNA damage. Although the phosphatidylinositol 3-kinases, DNA-PK (DNA-dependent proteinkinase), ATM (ataxia telangiectasiamutated), and ATR (ATM andRad3-related), have all been implicated in H2AX phosphorylation, the specific kinase involved has not yet been identified. To definitively identify the specific kinase(s) that phosphorylates H2AX in vivo, we have utilized DNA-PKcs−/− and Atm−/− cell lines and mouse embryonic fibroblasts. We find that H2AX phosphorylation and nuclear focus formation are normal in DNA-PKcs−/− cells and severely compromised in Atm−/− cells. We also find that ATM can phosphorylate H2AX in vitro and that ectopic expression of ATM in Atm−/− fibroblasts restores H2AX phosphorylation in vivo. The minimal H2AX phosphorylation in Atm−/− fibroblasts can be abolished by low concentrations of wortmannin suggesting that DNA-PK, rather than ATR, is responsible for low levels of H2AX phosphorylation in the absence of ATM. Our results clearly establish ATM as the major kinase involved in the phosphorylation of H2AX and suggest that ATM is one of the earliest kinases to be activated in the cellular response to double-strand breaks. double-strand break ionizing radiation phosphatidylinositol mouse embryonic fibroblasts DNA double-strand breaks (DSBs)1 are probably the most dangerous of the many different types of DNA damage that occur within the cell. DSBs are generated by exogenous agents such as ionizing radiation (IR) or by endogenously generated reactive oxygen species and occur as intermediates during meiotic and V(D)J recombination (1Khanna K.K. Jackson S.P. Nat. Genet. 2001; 27: 247-254Crossref PubMed Scopus (1867) Google Scholar). A very early step in the cellular response to DSBs is the phosphorylation of a histone H2A variant, H2AX, at the sites of DNA damage (2Modesti M. Kanaar R. Curr. Biol. 2001; 11: R229-R232Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). H2AX is rapidly phosphorylated (within seconds) at serine 139 when DSBs are introduced into mammalian cells (3Rogakou 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 (4049) Google Scholar) resulting in discrete γ-H2AX (phosphorylated-H2AX) foci at the DNA damage sites (4Rogakou E.P. Boon C. Redon C. Bonner W.M. J. Cell Biol. 1999; 146: 905-915Crossref PubMed Scopus (1931) Google Scholar). In experiments involving the use of “laser scissors” to introduce breaks into living cells, γ-H2AX foci localized specifically with the laser path through the cell nuclei clearly demonstrating that H2AX phosphorylation is specific to the sites of DNA damage (4Rogakou E.P. Boon C. Redon C. Bonner W.M. J. Cell Biol. 1999; 146: 905-915Crossref PubMed Scopus (1931) Google Scholar, 5Paull T.T. Rogakou E.P. Yamazaki V. Kirchgessner C.U. Gellert M. Bonner W.M. Curr. Biol. 2000; 10: 886-895Abstract Full Text Full Text PDF PubMed Scopus (1661) Google Scholar). H2AX phosphorylation also appears to be a general cellular response to processes involving DSB intermediates including V(D)J recombination in lymphoid cells (6Chen H.T. Bhandoola A. Difilippantonio M.J. Zhu J. Brown M.J. Tai X. Rogakou E.P. Brotz T.M. Bonner W.M. Ried T. Nussenzweig A. Science (Wash. D. C.). 2000; 290: 1962-1964Crossref PubMed Scopus (283) Google Scholar) and meiotic recombination in mice (7Mahadevaiah S.K. Turner J.M.A. Baudat F. Rogakou E.P. de Boer P. Blanco-Rodriguez J. Jasin M. Keeney S. Bonner W.M. Burgoyne P.S. Nat. Genet. 2001; 27: 271-276Crossref PubMed Scopus (685) Google Scholar). Phosphorylation of yeast H2A at serine 129 (homologous to serine 139 of mammalian H2AX) causes chromatin decondensation and is required for efficient DNA double-strand break repair (8Downs J.A. Lowndes N.F. Jackson S.P. Nature. 2000; 408: 1001-1004Crossref PubMed Scopus (529) Google Scholar). In mammals, phosphorylation of H2AX appears to play a critical role in the recruitment of repair or damage-signaling factors to the sites of DNA damage (5Paull T.T. Rogakou E.P. Yamazaki V. Kirchgessner C.U. Gellert M. Bonner W.M. Curr. Biol. 2000; 10: 886-895Abstract Full Text Full Text PDF PubMed Scopus (1661) Google Scholar, 9Rappold I. Iwabuchi K. Date T. Chen J. J. Cell Biol. 2001; 153: 613-620Crossref PubMed Scopus (396) Google Scholar). As H2AX phosphorylation plays a very early and important role in the cellular response to DNA double-strand breaks, it is important to specifically identify the kinase(s) involved in this event. Members of the PI 3-kinase family, including DNA-PK (DNA-dependent proteinkinase), ATM (ataxia telangiectasiamutated), and ATR (ATM andRad3-related), are involved in the responses of mammalian cells to DSBs (10Durocher D. Jackson S.P. Curr. Opin. Cell Biol. 2001; 13: 225-231Crossref PubMed Scopus (443) Google Scholar). γ-H2AX focus formation is inhibited by the PI 3-kinase inhibitor wortmannin, and H2AX phosphorylation is reduced in the DNA-PK-deficient human cell line M059J (5Paull T.T. Rogakou E.P. Yamazaki V. Kirchgessner C.U. Gellert M. Bonner W.M. Curr. Biol. 2000; 10: 886-895Abstract Full Text Full Text PDF PubMed Scopus (1661) Google Scholar). This led to the conclusion that DNA-PK and at least one other kinase, possibly ATM and/or ATR, can phosphorylate H2AX upon DNA damage (2Modesti M. Kanaar R. Curr. Biol. 2001; 11: R229-R232Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 5Paull T.T. Rogakou E.P. Yamazaki V. Kirchgessner C.U. Gellert M. Bonner W.M. Curr. Biol. 2000; 10: 886-895Abstract Full Text Full Text PDF PubMed Scopus (1661) Google Scholar, 10Durocher D. Jackson S.P. Curr. Opin. Cell Biol. 2001; 13: 225-231Crossref PubMed Scopus (443) Google Scholar, 11van Gent D.C. Hoeijmakers J.H. Kanaar R. Nat. Rev. Genet. 2001; 2: 196-206Crossref PubMed Scopus (938) Google Scholar). To unambiguously define the roles of ATM and DNA-PK in H2AX phosphorylation, we utilized cells derived from knockout mice for ATM or DNA-PKcs (the catalytic subunit of DNA-PK). We observed normal H2AX phosphorylation and γ-H2AX focus formation in irradiated fibroblasts derived from wild type or DNA-PKcs−/− mice. In contrast, H2AX phosphorylation and γ-H2AX focus formation were strikingly reduced to near background levels in fibroblasts from Atm−/− mice. Ectopic expression of ATM in Atm−/− cells restored H2AX phosphorylation. Moreover, we show that immunoprecipitated ATM can phosphorylate recombinant H2AX in vitro. These results indicate that ATM, not DNA-PK, is the major kinase responsible for modifying H2AX upon irradiation. The minimal H2AX phosphorylation in Atm−/− cells could be abolished by low concentrations of wortmannin suggesting that DNA-PK, rather than ATR, is responsible for low levels of γ-H2AX formation in the absence of ATM. Spontaneously immortalized mouse fibroblasts, derived from wild type, DNA-PKcs−/− (12Kurimasa A. Ouyang H. Dong L.J. Wang S. Li X. Cordon-Cardo C. Chen D.J. Li G.C. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1403-1408Crossref PubMed Scopus (158) Google Scholar), or Atm−/− mice (13Xu Y. Ashley T. Brainerd E.E. Bronson R.T. Meyn M.S. Baltimore D. Genes Dev. 1996; 10: 2411-2422Crossref PubMed Scopus (736) Google Scholar), were maintained in a humidified atmosphere with 5% CO2 in α-minimum Eagle’s medium supplemented with 10% fetal calf serum, 100 units/ml penicillin, and 100 μg/ml streptomycin. Mouse embryonic fibroblasts (MEFs) were isolated from 13.5-day-old embryos and maintained in α-minimum Eagle’s medium supplemented with 15% fetal calf serum. Cells were grown to about 70% confluence and irradiated with x-rays (300-kV, 12-mA, 0.5-mm Cu) at the rate of 5.5 gray/min to achieve a cumulative dose of 10 gray for all experiments unless otherwise mentioned. Cells were UV-irradiated at the rate of 0.15 J/m2/s to achieve a cumulative dose of 10 J/m2. Cells were harvested after 30 min, except in the case of time courses where they were harvested at time points ranging from 5 min to 8 h. Drug treatment of cells was carried out by the addition of the following DNA-damaging agents to the culture media for 1 h at the indicated concentrations: neocarzinostatin (0.2 μg/ml), bleomycin (50 μg/ml), etoposide (30 μg/ml), methyl methanesulfonate (50 μg/ml), and hydroxyurea (1 mm). Anti-γ-H2AX antibody was generated against a synthetic peptide consisting of the last nine amino acids of H2AX with phospho-Ser-139 as described before (3Rogakou 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 (4049) Google Scholar). SDS extracts for Western blotting were prepared from mock-irradiated or irradiated cells as described previously (14D'Anna J.A. Valdez J.G. Habbersett R.C. Crissman H.A. Radiat. Res. 1997; 148: 260-271Crossref PubMed Scopus (31) Google Scholar). The antibodies used for Western blotting are anti-γ-H2AX, anti-H2A (H-124; Santa Cruz Biotechnology Inc., Santa Cruz, CA), and anti-ATM monoclonal antibody MAT3–4G10/8 (15Andegeko Y. Moyal L. Mitelman L. Tsarfaty I. Shiloh Y. Rotman G. J. Biol. Chem. 2001; 276: 38224-38230Abstract Full Text Full Text PDF PubMed Google Scholar). Transient transfection of exponentially growing Atm−/− spontaneously immortalized fibroblasts with the ATM cDNA expression vector pMAT1 (16Zhang N. Chen P. Khanna K.K. Scott S. Gatei M. Kozlov S. Watters D. Spring K. Yen T. Lavin M.F. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8021-8026Crossref PubMed Scopus (97) Google Scholar) was carried out using Superfect transfection reagent (Qiagen Inc., Valencia, CA) as per the manufacturer’s protocols. Immediately after transfection, cells were induced for ATM expression with 5 μm CdCl2 for 16 h and then mock-irradiated or irradiated as described above. ATM immunoprecipitations were carried out as described (17Ziv Y. Banin S. Lim D.S. Canman C.E. Kastan M.B. Shiloh Y. Chan D.W. Gately D.P. Urban S. Galloway A.M. Lees-Miller S.P. Yen T. Allalunis-Turner J. Methods Mol. Biol. 2000; 99: 99-108PubMed Google Scholar). Approximately 1 × 107spontaneously immortalized mouse fibroblasts were grown to 70% confluence, mock-irradiated or irradiated, harvested after 30 min, and lysed in fresh cold lysis buffer containing protease and phosphatase inhibitors. The lysate was cleared by centrifugation, and the supernatant was incubated with 10 μg of anti-ATM monoclonal antibody MAT3–4G10/8 (15Andegeko Y. Moyal L. Mitelman L. Tsarfaty I. Shiloh Y. Rotman G. J. Biol. Chem. 2001; 276: 38224-38230Abstract Full Text Full Text PDF PubMed Google Scholar) for 2 h at 4 °C followed by incubation with protein A/G-Sepharose beads for an additional 2 h. The beads were washed repeatedly with lysis buffer, once with high salt buffer, and twice with kinase buffer. The beads were then incubated in a kinase mix (20 μl of kinase buffer, 500 ng of recombinant H2AX (purified from bacteria), 2 μl of 100 μm ATP, and 10 μCi of γ[32P]ATP) at 30 °C for 10 min. After SDS-polyacrylamide gel electrophoresis, the reaction products were visualized by autoradiography. Spontaneously immortalized fibroblasts were grown on chamber slides to about 70% confluence and then mock-irradiated or irradiated and incubated for 30 min. Cells were fixed in 4% paraformaldehyde for 10 min, permeabilized for 10 min in 0.2% Triton X-100, and blocked in 10% normal goat serum for 1 h at room temperature. The slides were incubated with anti-γ-H2AX antibody for 1 h, washed in phosphate-buffered saline, and incubated with Alexa Fluor 488-conjugated goat anti-rabbit secondary antibody (Molecular Probes, Eugene, OR) for 1 h at room temperature. Cells were washed in phosphate-buffered saline and mounted using Vectashield mounting medium with 4,6 diamidino-2-phenylindole (Vector Laboratory, Burlingame, CA). Fluorescence images were captured using an Olympus BH2 epifluorescent microscope equipped with a CCD camera and Cytovision software (Applied Imaging, Santa Clara, CA). To allow direct comparisons, all the cells were irradiated and processed simultaneously, and all the images were obtained using the same parameters (brightness, contrast, etc.). To examine H2AX phosphorylation in mouse cells, a rabbit polyclonal antibody (anti-γ-H2AX) was generated against a synthetic phosphorylated polypeptide consisting of the last nine amino acids of H2AX with phospho-Ser-139 (CKATQAS(PO4)QEY). The purified anti-γ-H2AX antibody reacted specifically with the immunizing polypeptide (phosphorylated at serine 139) but not with the unphosphorylated peptide (CKATQASQEY) (Fig. 1 a). Thus, the anti-γ-H2AX antibody is immunoreactive only with H2AX specifically phosphorylated at serine 139. Spontaneously immortalized wild type mouse fibroblasts were mock-irradiated or irradiated with x-rays and harvested after 30 min, and H2AX phosphorylation was analyzed by Western blotting of SDS extracts with anti-γ-H2AX antibody. We observed significant phosphorylation of histone H2AX in response to ionizing radiation (Fig. 1 b). The observed phosphorylation was specific to serine 139 as no signal was detected in irradiated samples when the immunizing polypeptide (phosphorylated at serine 139) was used as competitor in Western blotting (data not shown). Significant phosphorylation of H2AX was also observed after treatment of cells with the DSB-inducing agents neocarzinostatin, bleomycin, and etoposide. In contrast, there was no increase in γ-H2AX formation when these cells were irradiated with UV rays or treated with the DNA-alkylating agent methyl methanesulfonate confirming that H2AX is phosphorylated at serine 139 specifically in response to DNA double-strand breaks. Low levels of H2AX phosphorylation were also observed in cells treated with the DNA replication inhibitor hydroxyurea. This is probably because cells treated with hydroxyurea accumulate DSBs because of replication fork collapse (18Saintigny Y. Delacote F. Vares G. Petitot F. Lambert S. Averbeck D. Lopez B.S. EMBO J. 2001; 20: 3861-3870Crossref PubMed Scopus (259) Google Scholar, 19Skog S. Heiden T. Eriksson S. Wallström B. Tribukait B. Anticancer Drugs. 1992; PubMed Scopus Google Scholar). As the PI 3-kinases, DNA-PK, ATM, and ATR, have all been implicated in H2AX phosphorylation (2Modesti M. Kanaar R. Curr. Biol. 2001; 11: R229-R232Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 5Paull T.T. Rogakou E.P. Yamazaki V. Kirchgessner C.U. Gellert M. Bonner W.M. Curr. Biol. 2000; 10: 886-895Abstract Full Text Full Text PDF PubMed Scopus (1661) Google D. Jackson S.P. Curr. Opin. Cell Biol. 2001; 13: 225-231Crossref PubMed Scopus (443) Google Scholar, 11van Gent D.C. Hoeijmakers J.H. Kanaar R. Nat. Rev. Genet. 2001; 2: 196-206Crossref PubMed Scopus (938) Google Scholar), we to of these kinases a major role in the The PI 3-kinase inhibitor wortmannin the kinase of ATM and DNA-PK in cells with at concentrations of about 5 R.T. Res. 1998; Google Scholar). The kinase of ATR is to this with at concentrations than 100 Spontaneously immortalized wild type mouse fibroblasts were treated with concentrations of wortmannin for 30 min, irradiated with harvested after 30 min, and analyzed by Western We that H2AX phosphorylation was inhibited by low concentrations of wortmannin that ATM and/or DNA-PK, but not ATR, is involved in this (Fig. 1 Spontaneously immortalized fibroblasts from wild type, or Atm−/− mice were mock-irradiated or irradiated, harvested at time points ranging from 5 min to 8 h, and for H2AX phosphorylation by Western H2AX phosphorylation in wild type and DNA-PKcs−/− cells very rapidly (within 5 and for about 2 h, with levels of phosphorylation observed at 30 min (Fig. 2 a). In contrast, we observed minimal H2AX phosphorylation in irradiated Atm−/− cells. Although we observed H2AX phosphorylation in DNA-PKcs−/− cells at 30 min γ-H2AX formation in Atm−/− cells was reduced to about 5% of that in wild type cells (Fig. 2 that ATM is the major kinase responsible for H2AX phosphorylation upon DNA damage. Atm−/− fibroblasts were treated with concentrations of wortmannin for 30 min, irradiated with harvested after 30 min, and analyzed by Western We that the minimal H2AX phosphorylation in Atm−/− cells was abolished by low concentrations of wortmannin (Fig. 2 As ATR is inhibited by high concentrations of wortmannin R.T. Res. 1998; Google Scholar), results suggest that DNA-PK, rather than ATR, is responsible for low levels of γ-H2AX formation in the absence of ATM. is that other in the Atm−/− cell line used could also be responsible for the of H2AX phosphorylation in these cells. H2AX phosphorylation in a of early or or H2AX phosphorylation was observed in irradiated (Fig. In contrast, very low levels of γ-H2AX formation was observed in Atm−/− confirming that ATM is required for H2AX phosphorylation in response to significant in H2AX phosphorylation was observed irradiated and (Fig. To that ATM is required in for H2AX phosphorylation, the ATM cDNA expression vector pMAT1 (16Zhang N. Chen P. Khanna K.K. Scott S. Gatei M. Kozlov S. Watters D. Spring K. Yen T. Lavin M.F. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8021-8026Crossref PubMed Scopus (97) Google Scholar) was into Atm−/− spontaneously immortalized fibroblasts. The ectopic expression of ATM in the cells in of H2AX phosphorylation upon (Fig. 2 and the other cells with the vector no increase in γ-H2AX formation (Fig. The ATM expression and H2AX phosphorylation that ATM is required in for γ-H2AX formation in response to ionizing To ATM can phosphorylate H2AX in ATM was immunoprecipitated from spontaneously immortalized wild type fibroblasts using an anti-ATM monoclonal antibody against a peptide of ATM (15Andegeko Y. Moyal L. Mitelman L. Tsarfaty I. Shiloh Y. Rotman G. J. Biol. Chem. 2001; 276: 38224-38230Abstract Full Text Full Text PDF PubMed Google Scholar). The immunoprecipitated ATM phosphorylated recombinant H2AX in vitro (Fig. of cells in a significant increase in H2AX phosphorylation (Fig. no ATM protein or kinase was detected when was with normal mouse or from Atm−/− fibroblasts (Fig. The in vitro phosphorylation of H2AX by ATM that ATM could phosphorylate H2AX within the cell in response to DNA damage. H2AX phosphorylation in response to DNA damage results in the formation of discrete γ-H2AX foci at the sites of DNA double-strand breaks (4Rogakou E.P. Boon C. Redon C. Bonner W.M. J. Cell Biol. 1999; 146: 905-915Crossref PubMed Scopus (1931) Google Scholar). To the of γ-H2AX focus formation in wild type, and Atm−/− spontaneously immortalized fibroblasts, these cells were irradiated and to for 30 min before and with anti-γ-H2AX antibody. We observed γ-H2AX focus formation upon of wild type and DNA-PKcs−/− cells (Fig. In contrast, focus formation was very in Atm−/− cells confirming that ATM is required for γ-H2AX focus formation at the sites of H2AX is rapidly phosphorylated at serine 139 in response to DNA double-strand breaks (3Rogakou 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 (4049) Google Scholar). The PI 3-kinases, DNA-PK, ATM, and ATR, have all been implicated in this (2Modesti M. Kanaar R. Curr. Biol. 2001; 11: R229-R232Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 5Paull T.T. Rogakou E.P. Yamazaki V. Kirchgessner C.U. Gellert M. Bonner W.M. Curr. Biol. 2000; 10: 886-895Abstract Full Text Full Text PDF PubMed Scopus (1661) Google Scholar, 10Durocher D. Jackson S.P. Curr. Opin. Cell Biol. 2001; 13: 225-231Crossref PubMed Scopus (443) Google Scholar, 11van Gent D.C. Hoeijmakers J.H. Kanaar R. Nat. Rev. Genet. 2001; 2: 196-206Crossref PubMed Scopus (938) Google Scholar). Although the of these kinases are in they have clearly in D. Jackson S.P. Curr. Opin. Cell Biol. 2001; 13: 225-231Crossref PubMed Scopus (443) Google Scholar). ATM phosphorylates and to cell DNA-PK is not required for of these processes S. A. G. Y. R. M. Crissman H.A. Ouyang H. Li G.C. Chen D.J. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, M.B. Lim D.S. Nat. Rev. Mol. Biol. 2000; PubMed Scopus Google Scholar). the other DNA-PK, ATM, be involved in the recruitment of and DNA to the sites of DSBs G.C. Jackson S.P. Genes Dev. 1999; 13: PubMed Scopus Google Scholar). is important to definitively the roles of these kinases in the phosphorylation of We that ATM can phosphorylate H2AX in vitro and that H2AX phosphorylation and γ-H2AX focus formation are severely compromised in Atm−/− cells. Ectopic expression of ATM this In contrast, these are normal in DNA-PKcs−/− cells. DNA-PK, but not ATR, be responsible for the minimal levels of H2AX phosphorylation in Atm−/− cells as this can be abolished by low concentrations of We also find that immunoprecipitated ATM can with recombinant H2AX in and J. and experiments be to examine formation H2AX and ATM in vivo. Our results establish that ATM is the major kinase responsible for histone H2AX phosphorylation in response to DNA double-strand breaks in fibroblasts. The reduced H2AX phosphorylation in M059J cells (5Paull T.T. Rogakou E.P. Yamazaki V. Kirchgessner C.U. Gellert M. Bonner W.M. Curr. Biol. 2000; 10: 886-895Abstract Full Text Full Text PDF PubMed Scopus (1661) Google Scholar) be because of the low levels of ATM in these cells B.S. Kirchgessner C.U. Radiat. Res. 2000; 153: PubMed Scopus Google Scholar, D.P. Chan Yen Mol. Biol. 1998; PubMed Scopus Google Scholar, D.W. Gately D.P. Urban S. Galloway A.M. Lees-Miller S.P. Yen T. Allalunis-Turner J. J. Radiat. Biol. 1998; PubMed Scopus Google Scholar) rather than because of the absence of ATM plays a role in the of in response to to repair of DNA of cell and cellular responses G. Shiloh Y. 1999; PubMed Scopus Google Scholar). As γ-H2AX focus formation is a very early within of DNA damage (3Rogakou 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 (4049) Google Scholar), results indicate that ATM is one of the earliest kinases to be activated in the cellular response to DNA double-strand breaks. the important role of ATM in chromatin is a that ATM is also required for the of histone is to to chromatin decondensation Wang Y. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). Our results are with a that a of nuclear ATM with γ-H2AX at the sites of DSBs in response to DNA damage (15Andegeko Y. Moyal L. Mitelman L. Tsarfaty I. Shiloh Y. Rotman G. J. Biol. Chem. 2001; 276: 38224-38230Abstract Full Text Full Text PDF PubMed Google Scholar). A very was the of and of γ-H2AX foci and ATM This that DNA by ATM and H2AX phosphorylation occur very rapidly after DNA and at the same rate In the of this suggest that once ATM is activated at a it could phosphorylate histone H2AX at the of the break to the cell that a DSB has This very early could then the recruitment of DNA repair or damage-signaling factors to the break by chromatin and/or direct of these factors with We are to Bonner of for the anti-γ-H2AX antibody used in Shiloh for anti-ATM and Lavin of for We for and and for the