Rapid Activation of ATR by Ionizing Radiation Requires ATM and Mre11

Abstract

The ataxia-telangiectasia-mutated (ATM) and ATM- and Rad3-related (ATR) protein kinases are crucial regulatory proteins in genotoxic stress response pathways that pause the cell cycle to permit DNA repair. Here we show that Chk1 phosphorylation in response to hydroxyurea and ultraviolet radiation is ATR-dependent and ATM- and Mre11-independent. In contrast, Chk1 phosphorylation in response to ionizing radiation (IR) is dependent on ATR, ATM, and Mre11. The ATR and ATM/Mre11 pathways are generally thought to be separate with ATM activation occurring early and ATR activation occurring as a late response to double strand breaks. However, we demonstrate that ATR is activated rapidly by IR, and ATM and Mre11 enhance ATR signaling. ATR-ATR-interacting protein recruitment to double strand breaks is less efficient in the absence of ATM and Mre11. Furthermore, IR-induced replication protein A foci formation is defective in ATM- and Mre11-deficient cells. Thus, ATM and Mre11 may stimulate the ATR signaling pathway by converting DNA damage generated by IR into structures that recruit and activate ATR. The ataxia-telangiectasia-mutated (ATM) and ATM- and Rad3-related (ATR) protein kinases are crucial regulatory proteins in genotoxic stress response pathways that pause the cell cycle to permit DNA repair. Here we show that Chk1 phosphorylation in response to hydroxyurea and ultraviolet radiation is ATR-dependent and ATM- and Mre11-independent. In contrast, Chk1 phosphorylation in response to ionizing radiation (IR) is dependent on ATR, ATM, and Mre11. The ATR and ATM/Mre11 pathways are generally thought to be separate with ATM activation occurring early and ATR activation occurring as a late response to double strand breaks. However, we demonstrate that ATR is activated rapidly by IR, and ATM and Mre11 enhance ATR signaling. ATR-ATR-interacting protein recruitment to double strand breaks is less efficient in the absence of ATM and Mre11. Furthermore, IR-induced replication protein A foci formation is defective in ATM- and Mre11-deficient cells. Thus, ATM and Mre11 may stimulate the ATR signaling pathway by converting DNA damage generated by IR into structures that recruit and activate ATR. Genome maintenance requires coordination of DNA repair with cell cycle events. ATM 3The abbreviations used are: ATM, ataxia-telangiectasia-mutated; ATR, ATM and Rad3-related; ATRIP, ATR-interacting protein; ssDNA, single-stranded DNA; A-T, ataxia-telangiectasia; Chk1, checkpoint kinase 1; PBS, phosphate-buffered saline; RPA, replication protein A; Nbs1, Nijmegen breakage syndrome; MRN, Mre11-Rad50-Nbs1; IR, ionizing radiation; Gy, gray; siRNA, small interfering RNA; ssDNA, single-stranded DNA; DTT, dithiothreitol; PIPES, piperazine-N,N′-bis(2-ethanesulfonic acid). 3The abbreviations used are: ATM, ataxia-telangiectasia-mutated; ATR, ATM and Rad3-related; ATRIP, ATR-interacting protein; ssDNA, single-stranded DNA; A-T, ataxia-telangiectasia; Chk1, checkpoint kinase 1; PBS, phosphate-buffered saline; RPA, replication protein A; Nbs1, Nijmegen breakage syndrome; MRN, Mre11-Rad50-Nbs1; IR, ionizing radiation; Gy, gray; siRNA, small interfering RNA; ssDNA, single-stranded DNA; DTT, dithiothreitol; PIPES, piperazine-N,N′-bis(2-ethanesulfonic acid). and ATR, members of the phosphatidylinositol kinase-like kinase family, are crucial regulatory proteins in genotoxic stress response pathways. Ultimately, ATM/ATR signaling halts the cell cycle to permit DNA repair, or in situations where repair systems are overwhelmed they initiate apoptosis (1Bakkenist C.J. Kastan M.B. Cell. 2004; 118: 9-17Abstract Full Text Full Text PDF PubMed Scopus (414) Google Scholar, 2Shiloh Y. Curr. Opin. Genet. Dev. 2001; 11: 71-77Crossref PubMed Scopus (516) Google Scholar, 3Sancar A. Lindsey-Boltz L.A. Unsal-Kacmaz K. Linn S. Annu. Rev. Biochem. 2004; 73: 39-85Crossref PubMed Scopus (2455) Google Scholar). Although the ATM and ATR kinases phosphorylate some common substrates, they are differentially activated by distinct types of DNA damage. A primary function of the ATR signaling pathway is to monitor DNA replication. When problems arise, ATR phosphorylates and activates the checkpoint kinase (Chk1) as well as many other proteins (4Liu Q. Guntuku S. Cui X.S. Matsuoka S. Cortez D. Tamai K. Luo G. Carattini-Rivera S. DeMayo F. Bradley A. Donehower L.A. Elledge S.J. Genes Dev. 2000; 14: 1448-1459Crossref PubMed Scopus (191) Google Scholar, 5Zhao H. Piwnica-Worms H. Mol. Cell. Biol. 2001; 21: 4129-4139Crossref PubMed Scopus (845) Google Scholar). Replication surveillance by ATR is accomplished by the recruitment of ATR and its accessory protein ATRIP to regions of replication protein A (RPA)-coated single-stranded DNA (ssDNA) that are generated by decoupling of helicase and polymerase activities at damaged replication forks (6Cortez D. Guntuku S. Qin J. Elledge S.J. Science. 2001; 294: 1713-1716Crossref PubMed Scopus (737) Google Scholar, 7Zou L. Elledge S.J. Science. 2003; 300: 1542-1548Crossref PubMed Scopus (2000) Google Scholar, 8Byun T.S. Pacek M. Yee M.C. Walter J.C. Cimprich K.A. Genes Dev. 2005; 19: 1040-1052Crossref PubMed Scopus (559) Google Scholar). Direct binding of ATRIP to RPA-coated ssDNA is required for the stable recruitment of the ATR-ATRIP complex to damaged sites (7Zou L. Elledge S.J. Science. 2003; 300: 1542-1548Crossref PubMed Scopus (2000) Google Scholar, 9Ball H.L. Myers J.S. Cortez D. Mol. Biol. Cell. 2005; 16: 2372-2381Crossref PubMed Scopus (183) Google Scholar). However, there may be alternative mechanisms by which the ATR-ATRIP complex can recognize damaged DNA other than direct ATRIP-RPA-ssDNA binding (9Ball H.L. Myers J.S. Cortez D. Mol. Biol. Cell. 2005; 16: 2372-2381Crossref PubMed Scopus (183) Google Scholar, 10Rouse J. Jackson S.P. Mol. Cell. 2002; 9: 857-869Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar, 11Unsal-Kacmaz K. Makhov A.M. Griffith J.D. Sancar A. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 6673-6678Crossref PubMed Scopus (136) Google Scholar, 12Unsal-Kacmaz K. Sancar A. Mol. Cell. Biol. 2004; 24: 1292-1300Crossref PubMed Scopus (80) Google Scholar, 13Bomgarden R.D. Yean D. Yee M.C. Cimprich K.A. J. Biol. Chem. 2004; 279: 13346-13353Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 14Kim S.M. Kumagai A. Lee J. Dunphy W.G. J. Biol. Chem. 2005; 280: 38355-38364Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). In contrast, ATM primarily initiates cellular responses to double strand breaks and is recruited to sites of damage by the Mre11-Rad50-Nbs1 (MRN) complex (14Kim S.M. Kumagai A. Lee J. Dunphy W.G. J. Biol. Chem. 2005; 280: 38355-38364Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 15Falck J. Coates J. Jackson S.P. Nature. 2005; 434: 605-611Crossref PubMed Scopus (975) Google Scholar, 16Bakkenist C.J. Kastan M.B. Nature. 2003; 421: 499-506Crossref PubMed Scopus (2635) Google Scholar, 17Uziel T. Lerenthal Y. Moyal L. Andegeko Y. Mittelman L. Shiloh Y. EMBO J. 2003; 22: 5612-5621Crossref PubMed Scopus (823) Google Scholar). Unlike ATR, ATM is not an essential gene, and ATM deficiencies result in a human disease, ataxia-telangiectasia (A-T). Cells from A-T patients are sensitive to IR but not to UV exposure. These observations have led to the general idea that ATM and ATR function in parallel pathways where cellular response to replicative stress and UV radiation is attributed to ATR, whereas ATM mediates cellular responses to IR-induced double strand breaks. However, there are several indications that ATR can also respond to double strand breaks. In A-T cells, p53 phosphorylation in response to IR is delayed but not completely abrogated (18Canman C.E. Wolff A.C. Chen C.Y. Fornace Jr., A.J. Kastan M.B. Cancer Res. 1994; 54: 5054-5058PubMed Google Scholar, 19Lu X. Lane D.P. Cell. 1993; 75: 765-778Abstract Full Text PDF PubMed Scopus (772) Google Scholar), and ATR is selectively responsible for the late phase of p53 phosphorylation (20Tibbetts R.S. Brumbaugh K.M. Williams J.M. Sarkaria J.N. Cliby W.A. Shieh S.Y. Taya Y. Prives C. Abraham R.T. Genes Dev. 1999; 13: 152-157Crossref PubMed Scopus (850) Google Scholar). The ATR-ATRIP complex is required for maintaining the G2/M checkpoint in response to IR (6Cortez D. Guntuku S. Qin J. Elledge S.J. Science. 2001; 294: 1713-1716Crossref PubMed Scopus (737) Google Scholar, 21Cliby W.A. Roberts C.J. Cimprich K.A. Stringer C.M. Lamb J.R. Schreiber S.L. Friend S.H. EMBO J. 1998; 17: 159-169Crossref PubMed Scopus (478) Google Scholar, 22Xu X. Tsvetkov L.M. Stern D.F. Mol. Cell. Biol. 2002; 22: 4419-4432Crossref PubMed Scopus (156) Google Scholar, 23Brown E.J. Baltimore D. Genes Dev. 2003; 17: 615-628Crossref PubMed Scopus (400) Google Scholar). The ATR-ATRIP complex is also recruited to IR-induced intranuclear foci (9Ball H.L. Myers J.S. Cortez D. Mol. Biol. Cell. 2005; 16: 2372-2381Crossref PubMed Scopus (183) Google Scholar, 24Tibbetts R.S. Cortez D. Brumbaugh K.M. Scully R. Livingston D. Elledge S.J. Abraham R.T. Genes Dev. 2000; 14: 2989-3002Crossref PubMed Scopus (397) Google Scholar). Thus, ATR is activated by double strand breaks, although the prevailing model is that this activation may be a late response compared with ATM signaling. Here we show that Chk1 phosphorylation in response to IR is rapid and ATR-dependent. ATM- and Mre11-deficient cells display defects in ATR-dependent Chk1 phosphorylation. ATR-ATRIP as well as RPA recruitment to double strand breaks is reduced in ATM- and Mre11-deficient cells. Therefore, ATM and Mre11 stimulate ATR-dependent Chk1 phosphorylation by promoting the conversion of DNA damage generated by IR into structures that recruit the ATR-ATRIP complex. Cell Culture and siRNA—All cell lines were maintained in Dulbecco's modified Eagle's medium plus 7.5% fetal bovine serum. Transfections of siRNA were performed with Oligofectamine (Invitrogen). The 19-base pair siRNA target sequences used are as follows: ATR, CCUCCGUGAUGUUGCUUGA; ATM, GCCUCCAGGCAGAAAAAGA; ATRIP, GGUCCACAGAUUAUUAGAU; Mre11, GCUAAUGACUCUGAUGAUA; Rad50, GCUCAGAGAUUGUGAAAUG; and Nbs1, GAAGAAACGUGAACUCAAG. The 19 base-pair nonspecific control oligonucleotide target sequence is AUGAACGUGAAUUGCUCAA. All RNA oligonucleotides were purchased from Dharmacon (Lafayette, CO). Drug Treatment and DNA Damage—Hydroxyurea was purchased from Sigma (catalogue no. H8627), dissolved in water at 1 m, and stored frozen. Aphidicolin was purchased from Sigma (catalogue no. A0781), dissolved in dimethyl sulfoxide at 5 mg/ml, and stored at -80 degrees. Etoposide (4′-demethylepipodophyllotoxin 9-(4,6-O-ethylidene-β-d-glucopyranoside)) was purchased from Sigma (catalogue no. E1383), dissolved in dimethyl sulfoxide at 10 mg/ml, and stored at -20 degrees. Cells were treated with ionizing radiation from a 137Cs source at a dose rate of 1.8 Gy/min. UV radiation was administered with a Stratalinker (Stratagene) after cells were washed one time with PBS. Proteomics Analysis—S3 HeLa nuclear extracts were generated from untreated and hydroxyurea-treated cells as follows. Briefly, S3 HeLa cells were grown in spinner flasks in Joklik's modified Eagle's medium purchased from Irvine Scientific (Santa Ana, CA) supplemented with 5% newborn calf serum (Invitrogen), sodium bicarbonate (Sigma, catalogue no. S3817), and 1× antibiotic/antimycotic (Invitrogen). Cells were harvested by centrifugation and washed twice with PBS. Cells were resuspended in hypotonic buffer (10 mm Tris-HCl (pH 7.9), 1.5 mm MgCl2, 10 mm KCl, 0.5 mm DTT), incubated on ice for 10 min, and homogenized. Resulting nuclei were recovered by centrifugation and resuspended in low salt buffer (20 mm Tris-HCl (pH 7.9), 25% glycerol, 0.2 mm EDTA, 1.5 mm MgCl2, 0.02 m KCl, 0.2 mm phenylmethylsulfonyl fluoride, and 0.5 mm DTT) and extracted by the addition of high salt buffer (20 mm Tris-HCl (pH 7.9), 25% glycerol, 0.2 mm EDTA, 1.5 mm MgCl2, 1.2 m KCl, 0.2 mm phenylmethylsulfonyl fluoride, and 0.5 mm DTT). The resulting extract was dialyzed in dialysis buffer (20 mm Tris-HCl (pH 7.9), 20% glycerol, 0.1 m KCl, 0.2 mm EDTA, 0.2 mm phenylmethylsulfonyl fluoride, 0.5 mm DTT). ATRIP was immunopurified from these extracts as described previously (25Wang Y. Cortez D. Yazdi P. Neff N. Elledge S.J. Qin J. Genes Dev. 2000; 14: 927-939Crossref PubMed Scopus (94) Google Scholar) with the following modifications. Immunoprecipitants were washed three times in NET buffer (made by combining NETN buffer (20 mm Tris-HCl (pH 7.5), 0.1 m NaCl, 1 mm EDTA, 0.5% Igepal CA-630) with Tris-buffered saline (50 mm Tris-HCl (pH 7.5), 150 mm NaCl) in a 1:1 ratio). Proteins were eluted in 8 m urea under conditions that preferentially eluted ATRIP-interacting proteins but not the ATRIP antibody. As a result, ATRIP, although present, was most likely underrepresented in the eluate. Eluates were fractionated by SDS-PAGE, and associated proteins were identified by two-dimensional liquid chromatography coupled tandem mass spectroscopy. Antibodies and Immunoblotting—Cells were lysed in 20 mm Tris (pH 7.5), 250 mm NaCl, 0.5% Igepal CA-630, 0.5% Tween 20, 2.5 mm EGTA supplemented with 5 μg/ml aprotinin, 5 μg/ml leupeptin, 1 mm NaF, 20 mm β-glycerophosphate, 1 mm sodium vanadate, 1 mm dithiothreitol, and 1 mm phenylmethylsulfonyl fluoride. Lysates were cleared by centrifugation prior to protein concentration determination (Bio-Rad). The ATRIP antibody has been described previously (6Cortez D. Guntuku S. Qin J. Elledge S.J. Science. 2001; 294: 1713-1716Crossref PubMed Scopus (737) Google Scholar). Antibodies to Chk1 and ATR were purchased from Santa Cruz Biotechnology. Chk1 P-S317 and Chk1 P-S345 antibodies were purchased from Cell Signaling Technology (Beverly, MA). ATM, Nbs1, and Mre11 antibodies were purchased from Novus Biologicals (Littleton, CO). Rad50 antibody was purchased from GeneTex (San Antonio, TX), and ATM P-S1981 antibody was purchased from Rockland Immunochemicals (Gilbertsville, PA). Immunofluorescence—Immunofluorescence was performed by plating cells directly on glass coverslips. For quantitation of Mre11 and RPA foci in HeLa cells, cells were washed twice with PBS, pre-extracted by a 5-min incubation on ice in MTSB buffer (10 mm PIPES, 50 mm NaCl, 5 mm MgCl2, 5 mm EGTA, 100 mm sucrose, 0.2% Triton X-100) followed by a 2-min incubation on ice in CSB buffer (10 mm Tris-HCl (pH 7.4), 10 mm NaCl, 3 mm MgCl2 0.1% Tween 20), washed twice with PBS plus 0.1% Triton X-100, washed twice in PBS, and fixed in methanol prior to permeabilization in Triton X-100 solution. HeLa cells analyzed for ATRIP foci and all other cells were fixed with paraformaldehyde and permeabilized with Triton X-100 solution as described previously (25Wang Y. Cortez D. Yazdi P. Neff N. Elledge S.J. Qin J. Genes Dev. 2000; 14: 927-939Crossref PubMed Scopus (94) Google Scholar). Fluorescein isothiocyanate- and rhodamine red-X-conjugated secondary antibodies were obtained from Jackson Immunoresearch. Mre11 and ATM Are Required for ATR-dependent Chk1 Phosphorylation in Response to IR but Are Dispensable in Response to UV Radiation and Hydroxyurea—Mre11, Nbs1, and Rad50 were identified in an ATRIP complex using a proteomics screen to identify novel ATRIP-associated and of HeLa cell that there be an the and ATR-ATRIP However, the of the complex in ATR or ATRIP was small than and the of ATR-ATRIP complex in of the complex was Furthermore, the that in untreated cells was not by DNA and we were to a direct proteins not have been some of a the complex and ATR signaling T. C. L. M. EMBO J. 2005; 24: PubMed Scopus (156) Google Scholar, H. A. M. Mol. Genet. 2005; 14: PubMed Scopus Google Scholar, P. F. EMBO J. 2004; PubMed Scopus Google Scholar). Therefore, we to the of the complex in the ATR response to genotoxic RNA we the of Mre11 in ATR signaling to genotoxic stress in HeLa cells. HeLa cells, with nonspecific siRNA oligonucleotides or siRNA Mre11 or ATR were to hydroxyurea mm for 5 UV radiation (50 for or IR for 1 As we that ATR was required to phosphorylate Chk1 on in response to hydroxyurea and UV but Mre11 was also for the ATM kinase in the hydroxyurea and UV However, of Mre11, ATM, and ATR IR-induced Chk1 phosphorylation with ATR the most and the Mre11 and ATM defects The for ATM and ATR for Chk1 phosphorylation after IR one of three ATM and ATR may target ATR may be of or ATM may be of ATR. ATM and ATR Chk1 we that to other ATM substrates, ATM be required for the early response and ATR for the late response to However, ATR was essential for Chk1 phosphorylation at the time we were to the time from to cell was Furthermore, ATM and ATR were in parallel pathways the of ATM and ATR Chk1 phosphorylation. However, cells in which ATM and ATR were in Chk1 phosphorylation the by ATR These that ATM and ATR function in a common signaling pathway to phosphorylate Chk1 in response to The for ATM and ATR was not to Chk1 on phosphorylation as were also required for Chk1 phosphorylation with the ATM activation we for ATR in the activation of ATM and ATM phosphorylation in cells and Thus, these the model in which ATM of ATR but in response to double strand breaks. The ATM for Chk1 phosphorylation was also in A-T cells. A-T cells or A-T cells with the ATM were harvested at time after ionizing radiation and analyzed for Chk1 phosphorylation. these cells a in Chk1 whereas Chk1 phosphorylation was in A-T cells with ATM The of ATM and Mre11 in Chk1 phosphorylation to IR For there was Mre11 for Chk1 activation cells were treated with the DNA polymerase or the The for ATM and Mre11 was not to HeLa cells the in cells Furthermore, of the other members of the Rad50 and Nbs1, to Mre11 A and these that the ATM and complex may ATR activation but in response to ionizing Mre11 and ATM RPA and ATR-ATRIP in Response to activation is to the of ssDNA with the protein RPA D. Genes Dev. 2005; 19: PubMed Scopus Google Scholar). Therefore, we that ATM and Mre11 may be for converting double strand breaks generated by IR into the DNA structures that recruit and activate ATR. ATM and Mre11 were required to ssDNA at IR-induced double strand breaks, we RPA and ATRIP foci formation in HeLa cells with nonspecific siRNA oligonucleotides or siRNA Mre11 and cells were untreated or treated with 5 of IR and fixed for 1 after the exposure. ATRIP and RPA foci were by In and A-T cells, Mre11, ATRIP, and RPA were to in response to ionizing radiation A and and that the Mre11 foci rapidly than the ATRIP foci not A parallel in RPA and ATRIP foci formation was in ATM- and Mre11 cells of cells of ATM ATRIP foci and RPA of cells of Mre11 ATRIP foci and RPA whereas and of cells with nonspecific oligonucleotides ATRIP and RPA Therefore, ATM and Mre11 may enhance ATR-ATRIP recruitment to double strand breaks by the of RPA-coated in ATRIP and RPA foci in A-T cells were by with A-T or A-T cells Y. A. P. Shiloh Y. PubMed Scopus Google Scholar) were to 5 of IR and for with the antibodies 1 after exposure. ATRIP, and Mre11 foci are These also demonstrate the of these proteins at IR-induced quantitation of ATRIP and foci in A-T or A-T cells. that these were not to HeLa cells with siRNA RPA and ATRIP foci were also in A-T cells and A-T cells with with the in HeLa cells, A-T cells defects in ATRIP and RPA foci in response to IR to A-T cells with ATM of cells ATM ATRIP foci and RPA In contrast, and of A-T cells with ATM ATRIP and RPA In Mre11 foci formation of ATM as by in the of cells Mre11 foci in A-T and A-T cells. Therefore, there was in the of double strand breaks in the A-T or A-T cells. these that Mre11 and ATM to activation of ATR by promoting the of RPA-coated ssDNA and promoting the recruitment of ATR-ATRIP to the sites of double strand breaks. demonstrate that Chk1 is rapidly in response to IR in an ATR-dependent ATM- and Mre11-deficient cells display defects in Chk1 phosphorylation that the ATR, ATM, and Mre11 proteins function in a common signaling The for Mre11 and ATM is to IR-induced ATR-dependent Chk1 phosphorylation and in ATR-dependent phosphorylation of Chk1 in response to UV radiation and that replicative also that Chk1 is an ATR as Chk1 phosphorylation is abrogated in cells, which ATM, and of ATM and ATR not Chk1 phosphorylation that by ATR Thus, we have identified a novel for ATM of the ATR signaling pathway for the of Mre11 and ATM in the ATR-dependent response to IR is that are required for an ATM signaling Mre11 is required for ATM activation T. Lerenthal Y. Moyal L. Andegeko Y. Mittelman L. Shiloh Y. EMBO J. 2003; 22: 5612-5621Crossref PubMed Scopus (823) Google Scholar, Science. 2005; PubMed Scopus Google Scholar, C.E. Lee EMBO J. 2003; 22: PubMed Scopus Google Scholar). of ATM in the phosphorylation of several proteins at double strand sites RPA H. J. Y. G. Cancer Res. 2001; Google Scholar, Y. S.P. Res. 2004; PubMed Scopus Google Scholar), which can be for ATR recruitment activation and Chk1 phosphorylation. ATM with the Mre11 may be in double strand breaks into the structures that ATR signaling. of RPA and ATRIP is with this model we a in RPA and ATR-ATRIP recruitment to foci in ATM- and Mre11-deficient cells. the recruitment of the ATR to the of DNA damage generated by IR is to be and T. S.J. EMBO J. 2005; 24: PubMed Scopus Google Scholar). In a separate the of the complex in at double strand breaks in the is also T. Nature. 2005; PubMed Scopus Google Scholar). These observations and a model in which ATM and Mre11 are required to double strand breaks into ssDNA, RPA The generated by ATM and Mre11 ATR-ATRIP which in Chk1 phosphorylation and cell cycle checkpoint model a for ATM and ATR to initiate the G2/M checkpoint in response to IR as well as the for ATR to the checkpoint E.J. Baltimore D. Genes Dev. 2003; 17: 615-628Crossref PubMed Scopus (400) Google Scholar). In the absence of ATM and Mre11 ATR activation although with reduced model also the prevailing idea that ATR is required for the late response to IR with that rapid Chk1 activation by IR is ATR-dependent. The the late activation of ATR model from in A-T cells delayed phosphorylation of as p53 in response to IR ATM is (18Canman C.E. Wolff A.C. Chen C.Y. Fornace Jr., A.J. Kastan M.B. Cancer Res. 1994; 54: 5054-5058PubMed Google Scholar, 19Lu X. Lane D.P. Cell. 1993; 75: 765-778Abstract Full Text PDF PubMed Scopus (772) Google Scholar, R.S. Brumbaugh K.M. Williams J.M. Sarkaria J.N. Cliby W.A. Shieh S.Y. Taya Y. Prives C. Abraham R.T. Genes Dev. 1999; 13: 152-157Crossref PubMed Scopus (850) Google Scholar). that the of ATM in these delayed direct phosphorylation and ATR the of this A. J. C. J. J. Jackson S.P. Cell Biol. 2005; PubMed Scopus Google Scholar) with that ATM and Mre11 enhance IR-induced ATR-dependent Chk1 at in RPA and ATR recruitment to double strand breaks. be to ATM and Mre11 the RPA-coated Mre11 is in response to double strand breaks and this phosphorylation activates its K. M. D. M. D. J. Mol. Cell. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). ATM an to of ssDNA that recruit and activate ATR. ATM and Mre11 may recruit other that the double strand breaks to In have for double strand breaks are by the ATM and ATR checkpoint for The mass was performed in the proteomics with from and from in with