Ataxia Telangiectasia Mutated (ATM) and ATM and Rad3-related Protein Exhibit Selective Target Specificities in Response to Different Forms of DNA Damage

Abstract

The ataxia telangiectasia mutated (ATM) and ATR (ATM and Rad3-related) protein kinases exert cell cycle delay, in part, by phosphorylating Checkpoint kinase (Chk) 1, Chk2, and p53. It is well established that ATR is activated following UV light-induced DNA damage such as pyrimidine dimers and the 6-(1,2)-dihydro-2-oxo-4-pyrimidinyl-5-methyl-2,4-(1H,3H)-pyrimidinediones, whereas ATM is activated in response to double strand DNA breaks. Here we clarify the activation of these kinases in cells exposed to IR, UV, and hyperoxia, a condition of chronic oxidative stress resulting in clastogenic DNA damage. Phosphorylation on Chk1(Ser-345), Chk2(Thr-68), and p53(Ser-15) following oxidative damage by IR involved both ATM and ATR. In response to ultraviolet radiation-induced stalled replication forks, phosphorylation on Chk1 and p53 required ATR, whereas Chk2 required ATM. Cells exposed to hyperoxia exhibited growth delay in G1, S, and G2 that was disrupted by wortmannin. Consistent with ATM or ATR activation, hyperoxia induced wortmannin-sensitive phosphorylation of Chk1, Chk2, and p53. By using ATM- and ATR-defective cells, phosphorylation on Chk1, Chk2, and p53 was found to be ATM-dependent, whereas ATR also contributed to Chk1 phosphorylation. These data reveal activated ATM and ATR exhibit selective substrate specificity in response to different genotoxic agents. The ataxia telangiectasia mutated (ATM) and ATR (ATM and Rad3-related) protein kinases exert cell cycle delay, in part, by phosphorylating Checkpoint kinase (Chk) 1, Chk2, and p53. It is well established that ATR is activated following UV light-induced DNA damage such as pyrimidine dimers and the 6-(1,2)-dihydro-2-oxo-4-pyrimidinyl-5-methyl-2,4-(1H,3H)-pyrimidinediones, whereas ATM is activated in response to double strand DNA breaks. Here we clarify the activation of these kinases in cells exposed to IR, UV, and hyperoxia, a condition of chronic oxidative stress resulting in clastogenic DNA damage. Phosphorylation on Chk1(Ser-345), Chk2(Thr-68), and p53(Ser-15) following oxidative damage by IR involved both ATM and ATR. In response to ultraviolet radiation-induced stalled replication forks, phosphorylation on Chk1 and p53 required ATR, whereas Chk2 required ATM. Cells exposed to hyperoxia exhibited growth delay in G1, S, and G2 that was disrupted by wortmannin. Consistent with ATM or ATR activation, hyperoxia induced wortmannin-sensitive phosphorylation of Chk1, Chk2, and p53. By using ATM- and ATR-defective cells, phosphorylation on Chk1, Chk2, and p53 was found to be ATM-dependent, whereas ATR also contributed to Chk1 phosphorylation. These data reveal activated ATM and ATR exhibit selective substrate specificity in response to different genotoxic agents. Cell cycle checkpoints are initiated in an attempt to prevent the fixation of mutations from one cell generation to the next. These checkpoints become activated when cells are exposed to genotoxic agents or other adverse environmental conditions (1Zhou B.B. Elledge S.J. Nature. 2000; 408: 433-439Crossref PubMed Scopus (2637) Google Scholar). Although the pathways of halting cell cycle progression are clear, the mechanisms of activating these checkpoints immediately following DNA damage are poorly understood. The ataxiatelangiectasia mutated (ATM) 1The abbreviations used are: ATM, ataxia-telangiectasia mutated; ATR, ATM and Rad3-related; Chk, Checkpoint kinase; IR, ionizing radiation; PIKK, phosphoinositide-3 kinase-related kinases; ATR-IP, ATR-interacting protein; kd, kinase dead; ROS, reactive oxygen species. and ataxia-telangiectasia and Rad3-related (ATR) phosphoinositide 3-kinase-related kinases (PIKKs) are critical for transducing DNA damage signals to checkpoint control proteins (2Bakkenist C.J. Kastan M.B. Cell. 2004; 118: 9-17Abstract Full Text Full Text PDF PubMed Scopus (417) Google Scholar). ATM is essential for mediating checkpoint control in cells exposed to ionizing radiation and other agents that produce double strand breaks in DNA. Cells derived from AT patients exhibit increased chromosome breaks, defects in cell cycle checkpoints, and increased cell death when exposed to IR. Although ATM kinase activity increases upon binding DNA ends produced by endonuclease cleavage or IR exposure (3Suzuki K. Kodama S. Watanabe M. J. Biol. Chem. 1999; 274: 25571-25575Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 4Smith G.C. Cary R.B. Lakin N.D. Hann B.C. Teo S.H. Chen D.J. Jackson S.P. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11134-11139Crossref PubMed Scopus (146) Google Scholar), recent studies have suggested ATM does not directly associate with DNA lesions in vivo. Instead, the auxiliary proteins Mre11, Rad50, and Nbs1 of the MRN complex recruit and activate ATM at sites of DNA lesions (5Lee J.H. Paull T.T. Science. 2004; 304: 93-96Crossref PubMed Scopus (585) Google Scholar). ATM has also been shown to be stimulated by changes in chromatin structure caused by damage to DNA (6Bakkenist C.J. Kastan M.B. Nature. 2003; 421: 499-506Crossref PubMed Scopus (2691) Google Scholar). In contrast, ATR is activated by stalled replication forks and agents that produce bulky adducts, such as UV irradiation (7Guo Z. Kumagai A. Wang S.X. Dunphy W.G. Genes Dev. 2000; 14: 2745-2756Crossref PubMed Scopus (366) Google Scholar, 8Unsal-Kacmaz K. Makhov A.M. Griffith J.D. Sancar A. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 6673-6678Crossref PubMed Scopus (137) Google Scholar). ATR activity is dependent upon its association with the ATR-interacting protein (ATR-IP) (9Cortez D. Guntuku S. Qin J. Elledge S.J. Science. 2001; 294: 1713-1716Crossref PubMed Scopus (749) Google Scholar) that targets the ATR-ATR-IP heterodimer to single-stranded DNA (10Zou L. Elledge S.J. Science. 2003; 300: 1542-1548Crossref PubMed Scopus (2051) Google Scholar). Thus, ATM and ATR are recruited to different complexes following genotoxic stress. Activation of these kinases by damaged DNA results in the direct or indirect phosphorylation or activation of a number of downstream checkpoint controls, DNA repair, or apoptosis-promoting targets including p53, Chk1, Chk2, Brca-1, Rad9, and Rad17 (7Guo Z. Kumagai A. Wang S.X. Dunphy W.G. Genes Dev. 2000; 14: 2745-2756Crossref PubMed Scopus (366) Google Scholar, 11Hekmat-Nejad M. You Z. Yee M.C. Newport J.W. Cimprich K.A. Curr. Biol. 2000; 10: 1565-1573Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar, 12Nur E.K.A. Li T.K. Zhang A. Qi H. Hars E.S. Liu L.F. J. Biol. Chem. 2003; 278: 12475-12481Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 13Kurz E.U. Lees-Miller S.P. DNA Repair. 2004; 3: 889-900Crossref PubMed Scopus (397) Google Scholar). Of major concern to cell cycle control are the checkpoint kinases Chk1 and Cds1 (hereafter referred to as Chk2). Chk1 and Chk2 become phosphorylated at serine 345 (14Zhao H. Piwnica-Worms H. Mol. Cell. Biol. 2001; 21: 4129-4139Crossref PubMed Scopus (867) Google Scholar) and threonine 68 (15Matsuoka S. Rotman G. Ogawa A. Shiloh Y. Tamai K. Elledge S.J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10389-10394Crossref PubMed Scopus (694) Google Scholar) following genotoxic exposure, respectively. The roles of Chk1 and Chk2 in response to DNA damage are essential, as inhibition of the ATM/Chk2 pathway results in a loss of the G2/M checkpoint and increased IR sensitivity (16Zhou B.B. Chaturvedi P. Spring K. Scott S.P. Johanson R.A. Mishra R. Mattern M.R. Winkler J.D. Khanna K.K. J. Biol. Chem. 2000; 275: 10342-10348Abstract Full Text Full Text PDF PubMed Scopus (262) Google Scholar), whereas Chk2 mutation has been implicated in the cancer Li-Fraumeni syndrome (17Vahteristo P. Tamminen A. Karvinen P. Eerola H. Eklund C. Aaltonen L.A. Blomqvist C. Aittomaki K. Nevanlinna H. Cancer Res. 2001; 61: 5718-5722PubMed Google Scholar). ATM and ATR can also directly phosphorylate p53(Ser-15) (18Canman C.E. Lim D.S. Cimprich K.A. Taya Y. Tamai K. Sakaguchi K. Appella E. Kastan M.B. Siliciano J.D. Science. 1998; 281: 1677-1679Crossref PubMed Scopus (1705) Google Scholar, 19Tibbetts 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 (866) Google Scholar, 20Delia D. Mizutani S. Panigone S. Tagliabue E. Fontanella E. Asada M. Yamada T. Taya Y. Prudente S. Saviozzi S. Frati L. Pierotti M.A. Chessa L. Br. J. Cancer. 2000; 82: 1938-1945Crossref PubMed Google Scholar) resulting in G1/S checkpoint initiation and increased cell viability following genotoxic insult. p53 activation is crucial to many cellular processes including DNA repair, cell cycle arrest, and apoptosis (for review see Ref. 21Tokino T. Nakamura Y. Crit. Rev. Oncol. Hematol. 2000; 33: 1-6Crossref PubMed Scopus (143) Google Scholar). Thus, activation of the Chk1, Chk2, and p53 checkpoint proteins is necessary for maintaining genome stability and overall cellular viability following genotoxic stress. Whereas both ATM and ATR are able to target all three of these checkpoint proteins, it remains unclear why different genotoxic stresses result in their specific activation and ability to differentially phosphorylate downstream targets. For example, ATM is rapidly activated by IR and phosphorylates p53(Ser-15), whereas ATR is slowly activated and is involved in maintaining p53(Ser-15) phosphorylation (18Canman C.E. Lim D.S. Cimprich K.A. Taya Y. Tamai K. Sakaguchi K. Appella E. Kastan M.B. Siliciano J.D. Science. 1998; 281: 1677-1679Crossref PubMed Scopus (1705) Google Scholar, 19Tibbetts 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 (866) Google Scholar). However, in response to UV-induced damage, ATR phosphorylates p53(Ser-15), and ATM is not required (19Tibbetts 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 (866) Google Scholar, 20Delia D. Mizutani S. Panigone S. Tagliabue E. Fontanella E. Asada M. Yamada T. Taya Y. Prudente S. Saviozzi S. Frati L. Pierotti M.A. Chessa L. Br. J. Cancer. 2000; 82: 1938-1945Crossref PubMed Google Scholar). Original reports indicated that ATM and ATR phosphorylate Chk2 and Chk1, respectively, thereby restricting cross-talk between the PIKKs and their downstream targets (7Guo Z. Kumagai A. Wang S.X. Dunphy W.G. Genes Dev. 2000; 14: 2745-2756Crossref PubMed Scopus (366) Google Scholar, 14Zhao H. Piwnica-Worms H. Mol. Cell. Biol. 2001; 21: 4129-4139Crossref PubMed Scopus (867) Google Scholar, 22Liu 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 (195) Google Scholar). Recently, ATR-mediated phosphorylation of Chk1 in response to IR was reported (23Gatei M. Sloper K. Sorensen C. Syljuasen R. Falck J. Hobson K. Savage K. Lukas J. Zhou B.B. Bartek J. Khanna K.K. J. Biol. Chem. 2003; 278: 14806-14811Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar) suggesting that these two parallel damage response branches not be these that the ATM and ATR kinases to to of genotoxic stress. Although the of ATM and ATR in the ability of these kinases to complexes with auxiliary proteins at specific damaged For example, IR and double strand breaks with a number of whereas UV dimers resulting in stalled replication forks DNA The that stalled replication forks activate ATR the response to IR. Thus, ATM as the following double strand whereas ATR be activated when stalled replication forks replication of damaged DNA. replication stalled by bulky or strand breaks, ATR be activated to cell cycle In ATM and ATR have to that DNA replication does not adverse environmental IR or UV, exposure to hyperoxia genotoxic reactive oxygen the of and P. PubMed Scopus Google Scholar). of the chronic of exposure, the growth of hyperoxia to cellular and of critical required for cell G.C. Cancer Res. Google Scholar). Although it is well that hyperoxia and and DNA strand breaks H. PubMed Scopus Google Scholar, J.M. D.J. M.A. J. 2004; PubMed Scopus Google Scholar), the of checkpoint control has been exposure to IR, hyperoxia growth delay by p53(Ser-15) phosphorylation and of C.E. M.A. J. 2001; PubMed Google Scholar, C.E. R.A. M.A. J. 2003; Scholar, C.E. M.A. Sci. 2001; PubMed Scopus Google Scholar). However, IR, cells and in cells exposed to hyperoxia in to of DNA initiation and C.E. M.A. J. 2001; PubMed Google Scholar). growth in be to stalled replication forks that as cells attempt to oxidative lesions a recent that p53(Ser-15) phosphorylation required ATR of hyperoxia R. J. 2004; PubMed Scopus Google Scholar) a response of stalled replication the reported that ATM was not required for it is to be activated by oxidative stress. ATM and ATR are activated following IR, it remains that ATM is activated exposure and ATR is activated The of was to the roles of the ATM and ATR kinases to phosphorylate the checkpoint proteins Chk1, Chk2, and p53 in response to different that produce DNA By using and and cells with of the for ATM and ATR to phosphorylate these checkpoint proteins in response to IR, UV, and hyperoxia was studies reveal that ATM and ATR become activated following IR, UV, or hyperoxia exhibit roles in their ability to phosphorylate Chk1, Chk2, and p53. Cell cells from the and in with and cells with a in with to control and and for selective of the P. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar) and and respectively, from the Cell These cells at in with and and genotoxic cells on a on and to The cells with of and the exposure to with or hyperoxia in a was to the to The was and with a at a of for For ionizing radiation exposed to their using a radiation at a of For ultraviolet exposed to using a cells in for to genotoxic cells with with of and of by the The for in at and The protein was by and to The in for Cell Cell p53(Ser-15) Cell p53 or as a control the in the as or The Jackson the with specific by are Cell by of using for and was Cell has been shown to DNA and growth in all of the cell cycle with of C.E. M.A. J. 2001; PubMed Google Scholar, C.E. M.A. Sci. 2001; PubMed Scopus Google Scholar, M.A. J. Cell. 2002; PubMed Scopus Google Scholar). shown C.E. M.A. J. 2001; PubMed Google Scholar), cells in all of the cell cycle with a in In contrast, cells to exposure and in and G2 PIKKs required for growth arrest, and cells exposed to or hyperoxia for in of the wortmannin. disrupted cell cycle hyperoxia with of cells exposed to of the of cells in of the cell cycle that the of and cells in G2 and The of G2 cells with an in the of cells in in the of cells in be caused by of cells in G2 of the cell cycle by the of other checkpoint proteins such as M. M. J. C. M. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). pathways involved in cells of hyperoxia and on cell cycle in increased the of cells in G2/M of the cell cycle with the of cells that in G2/M hyperoxia increased the of cells in with increased the of cells in G2/M of the cell cycle with increased the of cells in with the of cells that in G2/M hyperoxia in a of hyperoxia and on cell cycle in increased the of cells in G2/M of the cell cycle with the of cells that in G2/M of the cell cycle with increased the of cells in G2/M of the cell cycle with the of cells that in G2/M of the cell cycle with in a Checkpoint a growth in has been to p53(Ser-15) phosphorylation and activation of the of on p53 in cells exposed to or hyperoxia p53(Ser-15) phosphorylation was not in However, p53(Ser-15) and p53 protein increased hyperoxia, with increased of phosphorylation of p53(Ser-15) and overall protein of both p53 and in a the checkpoint kinases and are also downstream targets of and in these of both and in to hyperoxia in increased of both and that by in a and also in cells exposed to or of hyperoxia and not in cells in However, phosphorylated and in cells exposed to hyperoxia, with by wortmannin. these data that chronic oxidative stress caused by hyperoxia phosphorylation of the checkpoint proteins p53, Chk1, and Chk2 a wortmannin-sensitive ATM in Checkpoint Phosphorylation and exposed to or hyperoxia for 1, or to ATM in checkpoint control in a chronic of oxidative stress. cells exposed to hyperoxia exhibited an increased of p53(Ser-15) phosphorylation with cells at and data not and Consistent with phosphorylation on serine p53 activity not J. 1999; PubMed Scopus Google Scholar), p53 was of ATM and phosphorylation also increased in hyperoxia, and their was in ATM is required for checkpoint control following oxidative it was of to the response of the ATM cells to oxidative damage caused by IR. Consistent with studies S. Y. Y. Lees-Miller S.P. Appella E. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar), of p53(Ser-15) phosphorylation following IR in whereas phosphorylation was in and phosphorylation was in cells, that ATM is required for the phosphorylation of these kinases as Thus, ATM is necessary for phosphorylation of checkpoint proteins following and chronic oxidative stress. stalled replication forks ATM, the cell exposed to UV UV exposure stimulated phosphorylation of p53, Chk1, and Chk2 phosphorylation in an These data that ATM the Chk2 response when stalled replication forks are of are produced in response to ultraviolet ATR in Checkpoint Phosphorylation has been shown to p53(Ser-15) phosphorylation following damage. cells are cells of a of ATR used to the of ATR in response to IR, UV, and hyperoxia P. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). of these cells a protein that ATR with that cells with protein In response to hyperoxia, p53(Ser-15) was was in the of phosphorylation between cells or not 1, or of Consistent with the overall of p53 protein increased in p53 protein was not by also phosphorylation of Chk1 and Chk2 1, or of phosphorylation was by in phosphorylation was of hyperoxia when was as by These results that Chk1 not p53 or Chk2 phosphorylation ATR studies that ATR is required for IR to phosphorylate p53, Chk1, and reported (19Tibbetts 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 (866) Google Scholar), p53(Ser-15) phosphorylation in cells exposed to IR In contrast, p53 and of IR induced and phosphorylation. the of phosphorylation was upon of IR in the of with phosphorylation of protein following stress. These data that phosphorylation of Chk1(Ser-345), Chk2(Thr-68), and p53(Ser-15) following IR ATR. ATR is also activated by UV radiation and is required for phosphorylation of p53 and Chk1 (7Guo Z. Kumagai A. Wang S.X. Dunphy W.G. Genes Dev. 2000; 14: 2745-2756Crossref PubMed Scopus (366) Google Scholar). was by cells to UV and for p53(Ser-15), Chk1(Ser-345), and phosphorylation reported (19Tibbetts 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 (866) Google Scholar), the phosphorylation of p53(Ser-15) following UV radiation p53 protein and also phosphorylated UV phosphorylation was by These data studies that UV phosphorylation of p53 and Chk1 not that different of genotoxic stress activate specific DNA damage DNA strand breaks caused by IR activate the ATM/Chk2 whereas UV lesions and in DNA replication activate Whereas ATM and ATR two parallel branches of the DNA is in the these pathways Chk1 and Chk2 By using a of chronic oxidative stress in DNA is damaged of exposure, and with IR and UV damage, we able to that the pathways with to the activation of Chk1 and Chk2 in these studies is that of the genotoxic ATM or ATR phosphorylated the checkpoint proteins p53, Chk1, and for all three of damage, phosphorylation of the downstream targets are not it is the for ATM or ATR that is the DNA lesions produced by these agents are we that selective activation of the kinases the of target specificity following major of is that the cell exposed to different well established of genotoxic thereby direct of the response to specific of damage. studies that whereas ATM and ATR are their specificity for downstream targets upon the of that substrate specificity is caused by the of ATM and ATR to different complexes as a result of specific of a in checkpoint is for of damage or all damage is a In response to IR, the ATM on and phosphorylation is required for and initiation of ATM activity other (6Bakkenist C.J. Kastan M.B. Nature. 2003; 421: 499-506Crossref PubMed Scopus (2691) Google Scholar). of IR or agents that chromatin DNA strand breaks also ATM resulting in the In response to damage, ATM and to that are to be sites of DNA damage. The MRN complex at double strand breaks is necessary for ATM activity of and Nbs1 result in an J.H. Curr. Cell Biol. 2000; PubMed Scopus Google Scholar, Paull T. M. J. Biol. 2004; Scopus Google Scholar). ATM in phosphorylates is essential for phosphorylation of Chk2 at sites of DNA In contrast, phosphorylation of p53, a not These that Nbs1 substrate specificity of ATM. ATM, ATR activity is not stimulated by DNA damage. Instead, ATR to is recruited to replication protein to single-stranded DNA (10Zou L. Elledge S.J. Science. 2003; 300: 1542-1548Crossref PubMed Scopus (2051) Google Scholar). kinase activity the of the cell complex and DNA different of bulky lesions the of single-stranded DNA remains auxiliary proteins involved in different lesions substrate specificity for the ATR-ATR-IP heterodimer to replication protein Nbs1 signals to Chk2 specificity for ATM. in was that ATM was required for phosphorylation of all three checkpoint proteins the of ATM in the response to oxidative stress in In contrast, ATR phosphorylated hyperoxia, whereas it stimulated phosphorylation of all three checkpoint proteins following IR. Although it has been that IR and hyperoxia are in that both produce DNA damage R. P. Science. PubMed Scopus (585) Google Scholar), studies that the cellular response to these stresses is cells to IR, and hyperoxia H. J. J. J. Biol. PubMed Scopus Google Scholar, H. P. Google Scholar, H. Mol. PubMed Scopus Google Scholar). a recent R. J. 2004; PubMed Scopus Google Scholar) has suggested that the damage response to hyperoxia be different from that by IR. These data that the DNA lesions produced by IR and hyperoxia with their damage are substrate specificity be of oxidative DNA strand breaks. In the of hyperoxia, also as DNA of oxidative stress. be to the specific lesions produced exposure, these data reveal kinases are recruited genotoxic The that ATR is not required for p53 and Chk2 phosphorylation hyperoxia, is required following is also with the that hyperoxia and radiation activate at be to the that IR and double strand DNA breaks with a of whereas hyperoxia as a clastogenic DNA strand breaks, with for why IR and hyperoxia produce DNA lesions be to the of by IR a direct of from an radiation J. Biol. PubMed Scopus Google Scholar), whereas exposure an of by the of J.D. J. Biol. Chem. Full Text PDF PubMed Google Scholar, J.D. PubMed Scopus Google Scholar). Thus, be in the of lesions that activation of ATM and ATR. is that cells exposed to radiation are whereas cells exposed to hyperoxia are damaged for cells are oxidative stress following be to cell cycle progression with cells are to stalled replication forks that activate ATR. studies the established activation of the and pathway in the response to UV damage (7Guo Z. Kumagai A. Wang S.X. Dunphy W.G. Genes Dev. 2000; 14: 2745-2756Crossref PubMed Scopus (366) Google Scholar, 19Tibbetts 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 (866) Google Scholar). The activity of the ATM to phosphorylate Chk2 following UV exposure is a and is in the that ATM activity following UV of repair, DNA replication protein binding to DNA in double strand repair, and UV-induced DNA damage in M.A. A. M. Y. M. A. 2002; PubMed Scopus Google Scholar, J. M.A. M. Khanna K.K. K. Mol. Biol. Cell. 2001; PubMed Scopus Google Scholar). The activity of ATM in response to UV be to oxidative damage induced by H. and Scholar). Although studies in the following UV exposure, we the damage response we DNA strand breaks as a result of or to DNA. these of damaged DNA as for the binding and activation of the ATM with established and the of ATM to phosphorylate Chk2 following UV damage. DNA of and cells that disrupted G2 hyperoxia However, DNA of and cells in cell hyperoxia not not cell cycle hyperoxia not shown by of p53, Chk1, and Chk2, of ATM or inhibition of ATR is not to the checkpoint protein phosphorylation. an of targets between ATM and ATR wortmannin-sensitive such as protein be to these checkpoint proteins are with the that wortmannin-sensitive kinases are required to the DNA lesions produced In the that different genotoxic stresses activate checkpoint PIKKs an to clarify why these kinases are selective for their It is well that the downstream targets Chk1, Chk2, and p53 are phosphorylated by ATM or ATR in response to UV and hyperoxia, whereas IR phosphorylation of all three targets both ATM and ATR. that response by IR to ATM and ATR be of the of DNA damage produced by whereas the by UV and hyperoxia of damage such as stalled replication forks and cellular respectively. an substrate specificity of these kinases is by the binding and activation of ATM ATR with auxiliary proteins at sites of DNA the activation of these kinases selective be critical for activating specific DNA ATM and ATR are proteins with proteins involved in substrate checkpoint control and DNA studies different lesions selective activation of PIKKs and clarify these the cellular response to DNA damage. The was in by from the of of the at for the ATR cell also of the of and at the of for in