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• W2109805150 abstract "ATR (ataxiatelangiectasia and Rad-3-related) is a protein kinase required for survival after DNA damage. A critical role for ATR has been hypothesized to be the regulation of p53 and other cell cycle checkpoints. ATR has been shown to phosphorylate p53 at Ser15, and this damage-induced phosphorylation is diminished by expression of a catalytically inactive (ATR-kd) mutant. p53 function could not be examined directly in prior studies of ATR, however, because p53 was mutant or because cells expressed the SV40 large T antigen that blocks p53 function. To test the interactions of ATR and p53 directly we generated human U2OS cell lines inducible for either wild-type or kinase-dead ATR that also have an intact p53 pathway. Indeed, ATR-kd expression sensitized these cells to DNA damage and caused a transient decrease in damage-induced serine 15 phosphorylation of p53. However, we found that the effects of ATR-kd expression do not result in blocking the response of p53 to DNA damage. Specifically, prior ATR-kd expression had no effect on DNA damage-induced p53 protein up-regulation, p53-DNA binding, p21 mRNA up-regulation, or G1 arrest. Instead of promoting survival via p53 regulation, we found that ATR protects cells by delaying the generation of mitotic phosphoproteins and inhibiting premature chromatin condensation after DNA damage or hydroxyurea. Although p53 inhibition (by E6 or MDM2 expression) had little effect on premature chromatin condensation, when combined with ATR-kd expression there was a marked loss of the replication checkpoint. We conclude that ATR and p53 can function independently but that loss of both leads to synergistic disruption of the replication checkpoint. ATR (ataxiatelangiectasia and Rad-3-related) is a protein kinase required for survival after DNA damage. A critical role for ATR has been hypothesized to be the regulation of p53 and other cell cycle checkpoints. ATR has been shown to phosphorylate p53 at Ser15, and this damage-induced phosphorylation is diminished by expression of a catalytically inactive (ATR-kd) mutant. p53 function could not be examined directly in prior studies of ATR, however, because p53 was mutant or because cells expressed the SV40 large T antigen that blocks p53 function. To test the interactions of ATR and p53 directly we generated human U2OS cell lines inducible for either wild-type or kinase-dead ATR that also have an intact p53 pathway. Indeed, ATR-kd expression sensitized these cells to DNA damage and caused a transient decrease in damage-induced serine 15 phosphorylation of p53. However, we found that the effects of ATR-kd expression do not result in blocking the response of p53 to DNA damage. Specifically, prior ATR-kd expression had no effect on DNA damage-induced p53 protein up-regulation, p53-DNA binding, p21 mRNA up-regulation, or G1 arrest. Instead of promoting survival via p53 regulation, we found that ATR protects cells by delaying the generation of mitotic phosphoproteins and inhibiting premature chromatin condensation after DNA damage or hydroxyurea. Although p53 inhibition (by E6 or MDM2 expression) had little effect on premature chromatin condensation, when combined with ATR-kd expression there was a marked loss of the replication checkpoint. We conclude that ATR and p53 can function independently but that loss of both leads to synergistic disruption of the replication checkpoint. ionizing radiation green fluorescent protein premature chromatin condensation The response to DNA damage is a complex process that is crucial in maintaining the fidelity of the genome amid diverse stresses. A conserved and critical aspect of this response is cell cycle arrest immediately following damage, which allows for DNA repair prior to progression. The mechanisms of arrest following DNA damage are becoming increasingly well understood and in many cases involve a member of a conserved family of very large protein kinases called the phosphatidyl inositol kinase-related kinases (1Keith C.T. Schreiber S.L. Science. 1995; 270: 50-51Crossref PubMed Scopus (442) Google Scholar, 2Zhou B.B. Elledge S.J. Nature. 2000; 408: 433-439Crossref PubMed Scopus (2618) Google Scholar). In yeast, the Rad-3 (Schizosaccharomyces pombe) or Mec1 (Saccharomyces cerevisiae) family members are essential protein kinases that have been shown to be required for the response to diverse stresses including ultraviolet light, ionizing radiation, and hydroxyurea (3Jimenez G. Yucel J. Rowley R. Subramani S. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4952-4956Crossref PubMed Scopus (110) Google Scholar, 4Weinert T.A. Kiser G.L. Hartwell L.H. Genes Dev. 1994; 8: 652-665Crossref PubMed Scopus (668) Google Scholar). In humans, deficiency in ATM function leads to a complex phenotype including extreme sensitivity to ionizing radiation (IR),1 loss of p53 activation after IR, IR-resistant DNA synthesis, insulin resistance, chromosomal instability, loss of cerebellar neurons leading to ataxia, and development of lymphoid malignancies (5Savitsky K. Bar-Shira A. Gilad S. Rotman G. Ziv Y. Vanagaite L. Tagle D.A. Smith S. Uziel T. Sfez S. et al.Science. 1995; 268 (7): 1749-1753Crossref PubMed Scopus (2356) Google Scholar). The development of ATM-deficient mice and the human disease ataxia telangiectasia have led to a greater understanding of the functions of the ATM protein kinase.The role of ATR (ataxiatelangiectasia and Rad-3-related) has remained more mysterious than ATM because the ATR −/− mouse is early embryonic lethal (6Brown E.J. Baltimore D. Genes Dev. 2000; 14: 397-402PubMed Google Scholar, 7de Klein A. Muijtjens M. van Os R. Verhoeven Y. Smit B. Carr A.M. Lehmann A.R. Hoeijmakers J.H. Curr. Biol. 2000; 10: 479-482Abstract Full Text Full Text PDF PubMed Scopus (334) Google Scholar), and there is no specific small molecule inhibitor for ATR. In particular, the role of ATR in regulating p53 function has remained unclear. Several studies have demonstrated that in vitro ATR phosphorylates p53 on serine 15 (8–11), a site that is phosphorylated in response to DNA damage and plays a role in its transcriptional activation (12Dumaz N. Meek D.W. EMBO J. 1999; 18: 7002-7010Crossref PubMed Scopus (390) Google Scholar). In addition, Tibbetts et al. (13Tibbetts 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 (855) Google Scholar) demonstrated that in cells overexpressing ATR-kd, this serine 15 phosphorylation did not occur normally in response to damage by ultraviolet light or ionizing radiation, implicating ATR in the regulation of p53 in cells. It is also plausible that ATR may regulate p53 indirectly via Chk-1. Several studies have documented that ATR activates Chk-1 (14Hekmat-Nejad M. You Z. Yee M. Newport J.W. Cimprich K.A. Curr. Biol. 2000; 10: 1565-1573Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar, 15Guo Z. Kumagai A. Wang S.X. Dunphy W.G. Genes Dev. 2000; 14: 2745-2756Crossref PubMed Scopus (363) Google Scholar, 16Nghiem P. Park P.K. Kim Y. Vaziri C. Schreiber S.L. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 9092-9097Crossref PubMed Scopus (258) Google Scholar), and Chk-1 has been shown to phosphorylate p53 (17Shieh S.Y. Ahn J. Tamai K. Taya Y. Prives C. Genes Dev. 2000; 14: 289-300Crossref PubMed Google Scholar). Moreover, p53 levels are decreased when Chk-1 is inhibited, suggesting that p53 expression may be regulated by Chk-1 (17Shieh S.Y. Ahn J. Tamai K. Taya Y. Prives C. Genes Dev. 2000; 14: 289-300Crossref PubMed Google Scholar). Importantly, these prior studies have not been able to examine the role of ATR in regulating p53 function or the induction of a G1arrest because the p53 response was defective because of p53 mutations or expression of SV40 large T antigen, which binds p53 and blocks its function (18Sheppard H.M. Corneillie S.I. Espiritu C. Gatti A. Liu X. Mol. Cell. Biol. 1999; 19: 2746-2753Crossref PubMed Scopus (50) Google Scholar).To address the role of ATR in DNA damage-induced p53 activation and G1 arrest, we generated U2OS-derived stable cell lines that can inducibly overexpress either wild-type or kinase-dead ATR and that are functional in p53 and G1 arrest pathways. Here we report our results using these cell lines that demonstrate that ATR is not required for p53 activation or G1 arrest and that ATR works together with p53 in the replication checkpoint to block cells from prematurely condensing their chromatin.DISCUSSIONSeveral studies have shown that ATR can phosphorylate p53 on Ser15in vitro (8Hall-Jackson C.A. Cross D.A. Morrice N. Smythe C. Oncogene. 1999; 18: 6707-6713Crossref PubMed Scopus (193) Google Scholar, 9Lakin N.D. Hann B.C. Jackson S.P. Oncogene. 1999; 18: 3989-3995Crossref PubMed Scopus (111) Google Scholar, 10Kim S.T. Lim D.S. Canman C.E. Kastan M.B. J. Biol. Chem. 1999; 274: 37538-37543Abstract Full Text Full Text PDF PubMed Scopus (637) Google Scholar, 11Canman 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 (1693) Google Scholar) and in vivo(13Tibbetts 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 (855) Google Scholar), raising the possibility that ATR is involved in p53 regulation after DNA damage. Because of a variety of technical issues, prior studies could not examine the role of ATR in the functional regulation of p53. The data presented here are thus the first to examine the functional interactions of ATR and p53. Surprisingly, although expression of a catalytically inactive ATR caused marked sensitivity to DNA damage and a transient decrease in Ser15phosphorylation, ATR-kd had no effect on p53 activation by DNA damage. The simplest explanation for this finding is that ATR does not play a significant role in p53 functional activation. A related possibility is that there is functional overlap in p53 activation by multiple kinases such as ATM (26Kastan M.B. Zhan Q. el-Deiry W.S. Carrier F. Jacks T. Walsh W.V. Plunkett B.S. Vogelstein B. Fornace Jr., A.J. Cell. 1992; 71: 587-597Abstract Full Text PDF PubMed Scopus (2923) Google Scholar, 27Khanna K.K. Lavin M.F. Oncogene. 1993; 8: 3307-3312PubMed Google Scholar), p38 (28Bulavin D.V. Saito S. Hollander M.C. Sakaguchi K. Anderson C.W. Appella E. Fornace Jr., A.J. EMBO J. 1999; 18: 6845-6854Crossref PubMed Scopus (593) Google Scholar, 29Huang C. Ma W.Y. Maxiner A. Sun Y. Dong Z. J. Biol. Chem. 1999; 274: 12229-12235Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar), ATR, and possibly DNA-PK (10Kim S.T. Lim D.S. Canman C.E. Kastan M.B. J. Biol. Chem. 1999; 274: 37538-37543Abstract Full Text Full Text PDF PubMed Scopus (637) Google Scholar). In such a case, inhibition of one of these could be compensated by others. In either case, the data suggest that the major role of ATR in the response to DNA damage does not involve p53 regulation.Our data help in defining the relative roles of ATR and ATM in the response to DNA damage. There are many reasons to suspect that in some cases there may be functional overlap in the roles of ATR and ATM; both kinases phosphorylate p53 at Ser15 (11Canman 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 (1693) Google Scholar), they share overlapping substrate specificities (10Kim S.T. Lim D.S. Canman C.E. Kastan M.B. J. Biol. Chem. 1999; 274: 37538-37543Abstract Full Text Full Text PDF PubMed Scopus (637) Google Scholar), and ATR has been shown to rescue the radio-resistant DNA synthesis defect of ATM cells (22Cliby 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). Indeed, Zhou and Elledge (2Zhou B.B. Elledge S.J. Nature. 2000; 408: 433-439Crossref PubMed Scopus (2618) Google Scholar) have raised the concern that overexpression of ATR-kd may in fact be mediating some of its effects by inhibition of ATM function. There are several lines of evidence that suggest this is not the case: (a) ATM is well established as important in the activation of p53 and G1 arrest (especially following ionizing radiation) (26Kastan M.B. Zhan Q. el-Deiry W.S. Carrier F. Jacks T. Walsh W.V. Plunkett B.S. Vogelstein B. Fornace Jr., A.J. Cell. 1992; 71: 587-597Abstract Full Text PDF PubMed Scopus (2923) Google Scholar, 27Khanna K.K. Lavin M.F. Oncogene. 1993; 8: 3307-3312PubMed Google Scholar). In contrast, here we show that ATR-kd overexpression does not affect p53 activation by ultraviolet (Figs. Figure 3, Figure 4, Figure 5, Figure 6) or ionizing radiation (Fig. 3B and data not shown). (b) In a separate study (16Nghiem P. Park P.K. Kim Y. Vaziri C. Schreiber S.L. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 9092-9097Crossref PubMed Scopus (258) Google Scholar), we show that ATM does not play a role in the replication checkpoint that is likely a major function of ATR. In summary, it appears that in the p53/G1 checkpoint ATM plays a dominant role over ATR and that in the replication checkpoint, ATR is the relevant mediator rather than ATM. In the S phase (radio-resistant DNA synthesis) checkpoint, ATM has been well established to be involved through several mechanisms (30Falck J. Mailand N. Syljuasen R.G. Bartek J. Lukas J. Nature. 2001; 410: 842-847Crossref PubMed Scopus (865) Google Scholar, 31Lim D.S. Kim S.T. Xu B. Maser R.S. Lin J. Petrini J.H. Kastan M.B. Nature. 2000; 404: 613-617Crossref PubMed Scopus (671) Google Scholar), and ATR likely plays a role (22Cliby 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), perhaps through Chk-1 activation and cdc25A degradation (32Mailand N. Falck J. Lukas C. Syljuasen R.G. Welcker M. Bartek J. Lukas J. Science. 2000; 288: 1425-1429Crossref PubMed Scopus (641) Google Scholar).A further intriguing distinction between ATR and ATM lies in their mechanisms of activation; ATM kinase activity increases following DNA damage (11Canman 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 (1693) Google Scholar), whereas the kinase activity of ATR measured in vitro following in vivo DNA damage is unchanged (33Tibbetts 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 (398) Google Scholar). The most plausible model for ATR activation is that ATR co-localizes with its relevant substrates, such as BRCA-1, following DNA damage (33Tibbetts 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 (398) Google Scholar) and that this damage-induced co-localization involves DNA binding (14Hekmat-Nejad M. You Z. Yee M. Newport J.W. Cimprich K.A. Curr. Biol. 2000; 10: 1565-1573Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar), which has been shown to increase the kinase activity of ATR (8Hall-Jackson C.A. Cross D.A. Morrice N. Smythe C. Oncogene. 1999; 18: 6707-6713Crossref PubMed Scopus (193) Google Scholar, 9Lakin N.D. Hann B.C. Jackson S.P. Oncogene. 1999; 18: 3989-3995Crossref PubMed Scopus (111) Google Scholar). Given these observations of how ATR is activated in cells, we believe that expression of ATR-kd likely blocks endogenous ATR function by binding its activation partners into inactive complexes rather than by directly blocking the catalytic activity of endogenous ATR.Our studies of premature chromatin condensation are the first to investigate the functional interactions of ATR and p53 in the G1 and replication checkpoints, which we summarize in Fig.9. If ATR were required for p53 activation, one would expect that there would be little or no additional effect of p53 inhibition in cells expressing ATR-kd. In contrast we have found that inhibition of ATR and p53 function each independently sensitize cells to premature chromatin condensation and that the combination is more than additive (synergistic). Of note, our data suggest that ATR is more important (likely essential) in the replication checkpoint than p53 (see relative PCC rates for ATR-kd expression versus MDM2 or E6 expression). Portions of the pathway by which ATR likely functions in this checkpoint, via Chk-1 activation, have been relatively well characterized in Xenopus (14Hekmat-Nejad M. You Z. Yee M. Newport J.W. Cimprich K.A. Curr. Biol. 2000; 10: 1565-1573Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar, 15Guo Z. Kumagai A. Wang S.X. Dunphy W.G. Genes Dev. 2000; 14: 2745-2756Crossref PubMed Scopus (363) Google Scholar) and mammalian studies (16Nghiem P. Park P.K. Kim Y. Vaziri C. Schreiber S.L. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 9092-9097Crossref PubMed Scopus (258) Google Scholar,34Liu 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 (192) Google Scholar). In addition to the role of ATR in halting chromatin condensation before replication is complete, recent studies of Mec1 (the S. cerevisiae homolog of ATR) suggest ATR may also be required for progression of DNA replication (35Tercero J.A. Diffley J.F. Nature. 2001; 412: 553-557Crossref PubMed Scopus (556) Google Scholar), for stability of the replication fork (36Lopes M. Cotta-Ramusino C. Pellicioli A. Liberi G. Plevani P. Muzi-Falconi M. Newlon C.S. Foiani M. Nature. 2001; 412: 557-561Crossref PubMed Scopus (618) Google Scholar), and for blocking late origins of replication (35Tercero J.A. Diffley J.F. Nature. 2001; 412: 553-557Crossref PubMed Scopus (556) Google Scholar).Regarding the role of p53 in the replication checkpoint (25Taylor W.R. Stark G.R. Oncogene. 2001; 20: 1803-1815Crossref PubMed Scopus (1272) Google Scholar), there are a number of mechanisms by which this tumor suppressor can prevent cells from entering mitosis before DNA replication is complete: (a) p53 up-regulates 14-3-3-ς, which sequesters cdc25 outside the nucleus, blocking activation of mitosis promoting factor (37Chan T.A. Hermeking H. Lengauer C. Kinzler K.W. Vogelstein B. Nature. 1999; 401: 616-620Crossref PubMed Scopus (809) Google Scholar). (b) p53 is well established as a regulator of p21, which arrests the cell cycle in G1 and maintains a G2 arrest (38Bunz F. Dutriaux A. Lengauer C. Waldman T. Zhou S. Brown J.P. Sedivy J.M. Kinzler K.W. Vogelstein B. Science. 1998; 282: 1497-1501Crossref PubMed Scopus (2517) Google Scholar). (c) p53 induces Gadd45, which has been implicated in inducing the G2/M checkpoint (39Wang X.W. Zhan Q. Coursen J.D. Khan M.A. Kontny H.U. Yu L. Hollander M.C. O'Connor P.M. Fornace Jr., A.J. Harris C.C. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3706-3711Crossref PubMed Scopus (532) Google Scholar). (d) p53 diminishes expression of Cdc2 and cyclin B1 (25Taylor W.R. Stark G.R. Oncogene. 2001; 20: 1803-1815Crossref PubMed Scopus (1272) Google Scholar). All of these p53 effects slow the cell cycle, allow time for repair, and decrease the immediate need for an ATR-mediated arrest during DNA synthesis. In contrast, p53-mediated apoptosis is not playing a role in reducing the rate of PCC observed after ATR-kd expression; p53-null status and caspase inhibition have each been shown not to prevent PCC in ATR-deficient mouse embryos (6Brown E.J. Baltimore D. Genes Dev. 2000; 14: 397-402PubMed Google Scholar).Interestingly, we found that the cellular response to hydroxyurea (as well as to the more classical DNA damaging agent IR) was p53-dependent, as suggested by greater sensitivity to premature chromatin condensation upon HPV-E6 or MDM2 overexpression (Fig. 8). Others have reported that the response to hydroxyurea is dependent on p53 (40Taylor W.R. Agarwal M.L. Agarwal A. Stacey D.W. Stark G.R. Oncogene. 1999; 18: 283-295Crossref PubMed Scopus (63) Google Scholar), but this has not been universally observed and may depend on the cell line.An important implication of this work is that this level of ATR inhibition leaves p53 function fully intact, whereas the ATR/replication checkpoint is disabled. We have shown that defects in the G1 checkpoint (universally present in cancer cells) sensitize cells to lethal premature chromatin condensation by ATR inhibition (16Nghiem P. Park P.K. Kim Y. Vaziri C. Schreiber S.L. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 9092-9097Crossref PubMed Scopus (258) Google Scholar). This observation suggests that if a more potent and specific small molecule inhibitor of ATR can be discovered it may have cancer-selective properties by sensitizing these G1checkpoint-deficient cells to PCC. Our current results indicate that ATR inhibition does not disrupt p53 function and suggest that it may be important for an ATR inhibitor to be selective for ATR over ATM. This is because a small molecule that inhibited ATM function would cause the p53/G1 checkpoint of normal cells to be impaired, diminishing the selectivity of the inhibitor for cancer cells over normal cells. The response to DNA damage is a complex process that is crucial in maintaining the fidelity of the genome amid diverse stresses. A conserved and critical aspect of this response is cell cycle arrest immediately following damage, which allows for DNA repair prior to progression. The mechanisms of arrest following DNA damage are becoming increasingly well understood and in many cases involve a member of a conserved family of very large protein kinases called the phosphatidyl inositol kinase-related kinases (1Keith C.T. Schreiber S.L. Science. 1995; 270: 50-51Crossref PubMed Scopus (442) Google Scholar, 2Zhou B.B. Elledge S.J. Nature. 2000; 408: 433-439Crossref PubMed Scopus (2618) Google Scholar). In yeast, the Rad-3 (Schizosaccharomyces pombe) or Mec1 (Saccharomyces cerevisiae) family members are essential protein kinases that have been shown to be required for the response to diverse stresses including ultraviolet light, ionizing radiation, and hydroxyurea (3Jimenez G. Yucel J. Rowley R. Subramani S. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4952-4956Crossref PubMed Scopus (110) Google Scholar, 4Weinert T.A. Kiser G.L. Hartwell L.H. Genes Dev. 1994; 8: 652-665Crossref PubMed Scopus (668) Google Scholar). In humans, deficiency in ATM function leads to a complex phenotype including extreme sensitivity to ionizing radiation (IR),1 loss of p53 activation after IR, IR-resistant DNA synthesis, insulin resistance, chromosomal instability, loss of cerebellar neurons leading to ataxia, and development of lymphoid malignancies (5Savitsky K. Bar-Shira A. Gilad S. Rotman G. Ziv Y. Vanagaite L. Tagle D.A. Smith S. Uziel T. Sfez S. et al.Science. 1995; 268 (7): 1749-1753Crossref PubMed Scopus (2356) Google Scholar). The development of ATM-deficient mice and the human disease ataxia telangiectasia have led to a greater understanding of the functions of the ATM protein kinase. The role of ATR (ataxiatelangiectasia and Rad-3-related) has remained more mysterious than ATM because the ATR −/− mouse is early embryonic lethal (6Brown E.J. Baltimore D. Genes Dev. 2000; 14: 397-402PubMed Google Scholar, 7de Klein A. Muijtjens M. van Os R. Verhoeven Y. Smit B. Carr A.M. Lehmann A.R. Hoeijmakers J.H. Curr. Biol. 2000; 10: 479-482Abstract Full Text Full Text PDF PubMed Scopus (334) Google Scholar), and there is no specific small molecule inhibitor for ATR. In particular, the role of ATR in regulating p53 function has remained unclear. Several studies have demonstrated that in vitro ATR phosphorylates p53 on serine 15 (8–11), a site that is phosphorylated in response to DNA damage and plays a role in its transcriptional activation (12Dumaz N. Meek D.W. EMBO J. 1999; 18: 7002-7010Crossref PubMed Scopus (390) Google Scholar). In addition, Tibbetts et al. (13Tibbetts 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 (855) Google Scholar) demonstrated that in cells overexpressing ATR-kd, this serine 15 phosphorylation did not occur normally in response to damage by ultraviolet light or ionizing radiation, implicating ATR in the regulation of p53 in cells. It is also plausible that ATR may regulate p53 indirectly via Chk-1. Several studies have documented that ATR activates Chk-1 (14Hekmat-Nejad M. You Z. Yee M. Newport J.W. Cimprich K.A. Curr. Biol. 2000; 10: 1565-1573Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar, 15Guo Z. Kumagai A. Wang S.X. Dunphy W.G. Genes Dev. 2000; 14: 2745-2756Crossref PubMed Scopus (363) Google Scholar, 16Nghiem P. Park P.K. Kim Y. Vaziri C. Schreiber S.L. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 9092-9097Crossref PubMed Scopus (258) Google Scholar), and Chk-1 has been shown to phosphorylate p53 (17Shieh S.Y. Ahn J. Tamai K. Taya Y. Prives C. Genes Dev. 2000; 14: 289-300Crossref PubMed Google Scholar). Moreover, p53 levels are decreased when Chk-1 is inhibited, suggesting that p53 expression may be regulated by Chk-1 (17Shieh S.Y. Ahn J. Tamai K. Taya Y. Prives C. Genes Dev. 2000; 14: 289-300Crossref PubMed Google Scholar). Importantly, these prior studies have not been able to examine the role of ATR in regulating p53 function or the induction of a G1arrest because the p53 response was defective because of p53 mutations or expression of SV40 large T antigen, which binds p53 and blocks its function (18Sheppard H.M. Corneillie S.I. Espiritu C. Gatti A. Liu X. Mol. Cell. Biol. 1999; 19: 2746-2753Crossref PubMed Scopus (50) Google Scholar). To address the role of ATR in DNA damage-induced p53 activation and G1 arrest, we generated U2OS-derived stable cell lines that can inducibly overexpress either wild-type or kinase-dead ATR and that are functional in p53 and G1 arrest pathways. Here we report our results using these cell lines that demonstrate that ATR is not required for p53 activation or G1 arrest and that ATR works together with p53 in the replication checkpoint to block cells from prematurely condensing their chromatin. DISCUSSIONSeveral studies have shown that ATR can phosphorylate p53 on Ser15in vitro (8Hall-Jackson C.A. Cross D.A. Morrice N. Smythe C. Oncogene. 1999; 18: 6707-6713Crossref PubMed Scopus (193) Google Scholar, 9Lakin N.D. Hann B.C. Jackson S.P. Oncogene. 1999; 18: 3989-3995Crossref PubMed Scopus (111) Google Scholar, 10Kim S.T. Lim D.S. Canman C.E. Kastan M.B. J. Biol. Chem. 1999; 274: 37538-37543Abstract Full Text Full Text PDF PubMed Scopus (637) Google Scholar, 11Canman 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 (1693) Google Scholar) and in vivo(13Tibbetts 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 (855) Google Scholar), raising the possibility that ATR is involved in p53 regulation after DNA damage. Because of a variety of technical issues, prior studies could not examine the role of ATR in the functional regulation of p53. The data presented here are thus the first to examine the functional interactions of ATR and p53. Surprisingly, although expression of a catalytically inactive ATR caused marked sensitivity to DNA damage and a transient decrease in Ser15phosphorylation, ATR-kd had no effect on p53 activation by DNA damage. The simplest explanation for this finding is that ATR does not play a significant role in p53 functional activation. A related possibility is that there is functional overlap in p53 activation by multiple kinases such as ATM (26Kastan M.B. Zhan Q. el-Deiry W.S. Carrier F. Jacks T. Walsh W.V. Plunkett B.S. Vogelstein B. Fornace Jr., A.J. Cell. 1992; 71: 587-597Abstract Full Text PDF PubMed Scopus (2923) Google Scholar, 27Khanna K.K. Lavin M.F. Oncogene. 1993; 8: 3307-3312PubMed Google Scholar), p38 (28Bulavin D.V. Saito S. Hollander M.C. Sakaguchi K. Anderson C.W. Appella E. Fornace Jr., A.J. EMBO J. 1999; 18: 6845-6854Crossref PubMed Scopus (593) Google Scholar, 29Huang C. Ma W.Y. Maxiner A. Sun Y. Dong Z. J. Biol. Chem. 1999; 274: 12229-12235Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar), ATR, and possibly DNA-PK (10Kim S.T. Lim D.S. Canman C.E. Kastan M.B. J. Biol. Chem. 1999; 274: 37538-37543Abstract Full Text Full Text PDF PubMed Scopus (637) Google Scholar). In such a case, inhibition of one of these could be compensated by others. In either case, the data suggest that the major role of ATR in the response to DNA damage does not involve p53 regulation.Our data help in defining the relative roles of ATR and ATM in the response to DNA damage. There are many reasons to suspect that in some cases there may be functional overlap in the roles of ATR and ATM; both kinases phosphorylate p53 at Ser15 (11Canman 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 (1693) Google Scholar), they share overlapping substrate specificities (10Kim S.T. Lim D.S. Canman C.E. Kastan M.B. J. Biol. Chem. 1999; 274: 37538-37543Abstract Full Text Full Text PDF PubMed Scopus (637) Google Scholar), and ATR has been shown to rescue the radio-resistant DNA synthesis defect of ATM cells (22Cliby 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). Indeed, Zhou and Elledge (2Zhou B.B. Elledge S.J. Nature. 2000; 408: 433-439Crossref PubMed Scopus (2618) Google Scholar) have ra" @default.
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• W2109805150 title "ATR Is Not Required for p53 Activation but Synergizes with p53 in the Replication Checkpoint" @default.
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