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• W1855851918 abstract "•ATR protects a fraction of early S-phase cells from replication catastrophe•ATR suppresses DNA damage by coordinating RRM2 accumulation and origin firing•A DNA-PK-Chk1 pathway creates a threshold of tolerable ssDNA in ATRi-treated cells•ATRi-induced ssDNA may predict ATRi sensitivity in cancer cells The ATR-Chk1 pathway is critical for DNA damage responses and cell-cycle progression. Chk1 inhibition is more deleterious to cycling cells than ATR inhibition, raising questions about ATR and Chk1 functions in the absence of extrinsic replication stress. Here we show that a key role of ATR in S phase is to coordinate RRM2 accumulation and origin firing. ATR inhibitor (ATRi) induces massive ssDNA accumulation and replication catastrophe in a fraction of early S-phase cells. In other S-phase cells, however, ATRi induces moderate ssDNA and triggers a DNA-PK and Chk1-mediated backup pathway to suppress origin firing. The backup pathway creates a threshold such that ATRi selectively kills cells under high replication stress, whereas Chk1 inhibitor induces cell death at a lower threshold. The levels of ATRi-induced ssDNA correlate with ATRi sensitivity in a panel of cell lines, suggesting that ATRi-induced ssDNA could be predictive of ATRi sensitivity in cancer cells. The ATR-Chk1 pathway is critical for DNA damage responses and cell-cycle progression. Chk1 inhibition is more deleterious to cycling cells than ATR inhibition, raising questions about ATR and Chk1 functions in the absence of extrinsic replication stress. Here we show that a key role of ATR in S phase is to coordinate RRM2 accumulation and origin firing. ATR inhibitor (ATRi) induces massive ssDNA accumulation and replication catastrophe in a fraction of early S-phase cells. In other S-phase cells, however, ATRi induces moderate ssDNA and triggers a DNA-PK and Chk1-mediated backup pathway to suppress origin firing. The backup pathway creates a threshold such that ATRi selectively kills cells under high replication stress, whereas Chk1 inhibitor induces cell death at a lower threshold. The levels of ATRi-induced ssDNA correlate with ATRi sensitivity in a panel of cell lines, suggesting that ATRi-induced ssDNA could be predictive of ATRi sensitivity in cancer cells. The ATR (ataxia telangiectasia-mutated [ATM] and rad3-related) kinase is a crucial safeguard of the genome (Ciccia and Elledge, 2010Ciccia A. Elledge S.J. The DNA damage response: making it safe to play with knives.Mol. Cell. 2010; 40: 179-204Abstract Full Text Full Text PDF PubMed Scopus (2925) Google Scholar, Cimprich and Cortez, 2008Cimprich K.A. Cortez D. ATR: an essential regulator of genome integrity.Nat. Rev. Mol. Cell Biol. 2008; 9: 616-627Crossref PubMed Scopus (1314) Google Scholar, Flynn and Zou, 2011Flynn R.L. Zou L. ATR: a master conductor of cellular responses to DNA replication stress.Trends Biochem. Sci. 2011; 36: 133-140Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar, Maréchal and Zou, 2013Maréchal A. Zou L. DNA damage sensing by the ATM and ATR kinases.Cold Spring Harb. Perspect. Biol. 2013; 5: a012716Crossref Scopus (813) Google Scholar). In response to a wide range of intrinsic and extrinsic genotoxic stress, ATR acts as a master regulator of DNA damage signaling to orchestrate DNA repair, DNA replication, and cell-cycle progression. In particular, when DNA replication is compromised, ATR plays a key role in stabilizing the genome (Zeman and Cimprich, 2014Zeman M.K. Cimprich K.A. Causes and consequences of replication stress.Nat. Cell Biol. 2014; 16: 2-9Crossref PubMed Scopus (1151) Google Scholar). Several recent studies have shed light on how ATR functions in cells challenged with replication inhibitors, such as hydroxurea (HU) and aphidicolin (APH) (Couch et al., 2013Couch F.B. Bansbach C.E. Driscoll R. Luzwick J.W. Glick G.G. Bétous R. Carroll C.M. Jung S.Y. Qin J. Cimprich K.A. Cortez D. ATR phosphorylates SMARCAL1 to prevent replication fork collapse.Genes Dev. 2013; 27: 1610-1623Crossref PubMed Scopus (267) Google Scholar, Ragland et al., 2013Ragland R.L. Patel S. Rivard R.S. Smith K. Peters A.A. Bielinsky A.K. Brown E.J. RNF4 and PLK1 are required for replication fork collapse in ATR-deficient cells.Genes Dev. 2013; 27: 2259-2273Crossref PubMed Scopus (74) Google Scholar, Toledo et al., 2013Toledo L.I. Altmeyer M. Rask M.B. Lukas C. Larsen D.H. Povlsen L.K. Bekker-Jensen S. Mailand N. Bartek J. Lukas J. ATR prohibits replication catastrophe by preventing global exhaustion of RPA.Cell. 2013; 155: 1088-1103Abstract Full Text Full Text PDF PubMed Scopus (538) Google Scholar). In the presence of high levels of extrinsic replication stress, ATR is critical for preventing excessive cleavage of replication forks by nucleases and replication catastrophe, a state in which replicating chromosomes undergo severe fragmentation (Couch et al., 2013Couch F.B. Bansbach C.E. Driscoll R. Luzwick J.W. Glick G.G. Bétous R. Carroll C.M. Jung S.Y. Qin J. Cimprich K.A. Cortez D. ATR phosphorylates SMARCAL1 to prevent replication fork collapse.Genes Dev. 2013; 27: 1610-1623Crossref PubMed Scopus (267) Google Scholar, Ragland et al., 2013Ragland R.L. Patel S. Rivard R.S. Smith K. Peters A.A. Bielinsky A.K. Brown E.J. RNF4 and PLK1 are required for replication fork collapse in ATR-deficient cells.Genes Dev. 2013; 27: 2259-2273Crossref PubMed Scopus (74) Google Scholar, Toledo et al., 2013Toledo L.I. Altmeyer M. Rask M.B. Lukas C. Larsen D.H. Povlsen L.K. Bekker-Jensen S. Mailand N. Bartek J. Lukas J. ATR prohibits replication catastrophe by preventing global exhaustion of RPA.Cell. 2013; 155: 1088-1103Abstract Full Text Full Text PDF PubMed Scopus (538) Google Scholar). Although these studies provided important insights into one of the pivotal roles of ATR, how ATR functions under physiological and pathological conditions in the absence of extrinsic replication stress is yet to be elucidated. ATR and its homologs in a number of organisms are critical for the survival of proliferating cells. In budding yeast, the ATR homolog Mec1 is essential for viability unless Sml1, a repressor of ribonucleotide reductase, is deleted (Zhao et al., 1998Zhao X. Muller E.G. Rothstein R. A suppressor of two essential checkpoint genes identifies a novel protein that negatively affects dNTP pools.Mol. Cell. 1998; 2: 329-340Abstract Full Text Full Text PDF PubMed Scopus (600) Google Scholar). In mouse and C. elegans, loss of ATR leads to embryonic lethality (Brown and Baltimore, 2000Brown E.J. Baltimore D. ATR disruption leads to chromosomal fragmentation and early embryonic lethality.Genes Dev. 2000; 14: 397-402PubMed Google Scholar, Garcia-Muse and Boulton, 2005Garcia-Muse T. Boulton S.J. Distinct modes of ATR activation after replication stress and DNA double-strand breaks in Caenorhabditis elegans.EMBO J. 2005; 24: 4345-4355Crossref PubMed Scopus (137) Google Scholar). Conditional deletion of ATR from the human colon cancer cell line HCT116 also leads to cell death (Cortez et al., 2001Cortez D. Guntuku S. Qin J. Elledge S.J. ATR and ATRIP: partners in checkpoint signaling.Science. 2001; 294: 1713-1716Crossref PubMed Scopus (747) Google Scholar). However, ATR homologs in some other organisms, such as fission yeast and Drosophila, are not essential for viability (Enoch et al., 1992Enoch T. Carr A.M. Nurse P. Fission yeast genes involved in coupling mitosis to completion of DNA replication.Genes Dev. 1992; 6: 2035-2046Crossref PubMed Scopus (307) Google Scholar, Laurençon et al., 2003Laurençon A. Purdy A. Sekelsky J. Hawley R.S. Su T.T. Phenotypic analysis of separation-of-function alleles of MEI-41, Drosophila ATM/ATR.Genetics. 2003; 164: 589-601PubMed Google Scholar). Interestingly, in mouse, the effects of ATR loss on proliferating cells are not uniform in cell populations. For example, deletion of ATR in cells from blastocyosts resulted in different levels of genomic instability, ranging from a few DNA breaks to severe chromosomal fragmentation (Brown and Baltimore, 2000Brown E.J. Baltimore D. ATR disruption leads to chromosomal fragmentation and early embryonic lethality.Genes Dev. 2000; 14: 397-402PubMed Google Scholar). Deletion of ATR during nervous system development induced cell death only in specific progenitor cells (Lee et al., 2012Lee Y. Shull E.R. Frappart P.O. Katyal S. Enriquez-Rios V. Zhao J. Russell H.R. Brown E.J. McKinnon P.J. ATR maintains select progenitors during nervous system development.EMBO J. 2012; 31: 1177-1189Crossref PubMed Scopus (62) Google Scholar). These observations raise an important question as to why some proliferating cells are more dependent on ATR than others. How ATR functions during S phase is still poorly understood. During the response to DNA damage or replication stress, ATR phosphorylates and activates its effector kinase Chk1 (Liu et al., 2000Liu Q. Guntuku S. Cui X.S. Matsuoka S. Cortez D. Tamai K. Luo G. Carattini-Rivera S. DeMayo F. Bradley A. et al.Chk1 is an essential kinase that is regulated by Atr and required for the G(2)/M DNA damage checkpoint.Genes Dev. 2000; 14: 1448-1459Crossref PubMed Scopus (193) Google Scholar). It has been long believed that ATR and Chk1 function as a kinase cascade. Like ATR, Chk1 is critical for genomic stability during DNA replication (Forment et al., 2011Forment J.V. Blasius M. Guerini I. Jackson S.P. Structure-specific DNA endonuclease Mus81/Eme1 generates DNA damage caused by Chk1 inactivation.PLoS ONE. 2011; 6: e23517Crossref PubMed Scopus (85) Google Scholar, Petermann et al., 2008Petermann E. Helleday T. Caldecott K.W. Claspin promotes normal replication fork rates in human cells.Mol. Biol. Cell. 2008; 19: 2373-2378Crossref PubMed Scopus (89) Google Scholar, Petermann et al., 2010Petermann E. Woodcock M. Helleday T. Chk1 promotes replication fork progression by controlling replication initiation.Proc. Natl. Acad. Sci. USA. 2010; 107: 16090-16095Crossref PubMed Scopus (204) Google Scholar, Syljuåsen et al., 2005Syljuåsen R.G. Sørensen C.S. Hansen L.T. Fugger K. Lundin C. Johansson F. Helleday T. Sehested M. Lukas J. Bartek J. Inhibition of human Chk1 causes increased initiation of DNA replication, phosphorylation of ATR targets, and DNA breakage.Mol. Cell. Biol. 2005; 25: 3553-3562Crossref PubMed Scopus (445) Google Scholar). Both ATR and Chk1 have been implicated in the regulation of origin firing, even in the absence of extrinsic stress (Couch et al., 2013Couch F.B. Bansbach C.E. Driscoll R. Luzwick J.W. Glick G.G. Bétous R. Carroll C.M. Jung S.Y. Qin J. Cimprich K.A. Cortez D. ATR phosphorylates SMARCAL1 to prevent replication fork collapse.Genes Dev. 2013; 27: 1610-1623Crossref PubMed Scopus (267) Google Scholar, Eykelenboom et al., 2013Eykelenboom J.K. Harte E.C. Canavan L. Pastor-Peidro A. Calvo-Asensio I. Llorens-Agost M. Lowndes N.F. ATR activates the S-M checkpoint during unperturbed growth to ensure sufficient replication prior to mitotic onset.Cell Rep. 2013; 5: 1095-1107Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, Maya-Mendoza et al., 2007Maya-Mendoza A. Petermann E. Gillespie D.A. Caldecott K.W. Jackson D.A. Chk1 regulates the density of active replication origins during the vertebrate S phase.EMBO J. 2007; 26: 2719-2731Crossref PubMed Scopus (196) Google Scholar, Petermann et al., 2010Petermann E. Woodcock M. Helleday T. Chk1 promotes replication fork progression by controlling replication initiation.Proc. Natl. Acad. Sci. USA. 2010; 107: 16090-16095Crossref PubMed Scopus (204) Google Scholar, Shechter et al., 2004Shechter D. Costanzo V. Gautier J. ATR and ATM regulate the timing of DNA replication origin firing.Nat. Cell Biol. 2004; 6: 648-655Crossref PubMed Scopus (305) Google Scholar). However, a recent study reported unexpected differences between the effects of ATR inhibitor (ATRi) and Chk1 inhibitor (Chk1i) on cycling cells (Toledo et al., 2011Toledo L.I. Murga M. Zur R. Soria R. Rodriguez A. Martinez S. Oyarzabal J. Pastor J. Bischoff J.R. Fernandez-Capetillo O. A cell-based screen identifies ATR inhibitors with synthetic lethal properties for cancer-associated mutations.Nat. Struct. Mol. Biol. 2011; 18: 721-727Crossref PubMed Scopus (374) Google Scholar). Whereas Chk1i induced massive γH2AX accumulation in a large fraction of U2OS cells, ATRi only induced γH2AX in a small fraction of the cells. This result raises the possibility that ATR and Chk1 may not always function as a linear pathway, and it prompts a question about how ATR and Chk1 function in concert during DNA replication. While both ATR and Chk1 are important in cycling cells, the nature of the intrinsic stress that they deal with remains enigmatic. Interestingly, certain proliferation-promoting oncogenic events, such as Ras activation and Myc overexpression, render cancer cells sensitive to ATR suppression (Gilad et al., 2010Gilad O. Nabet B.Y. Ragland R.L. Schoppy D.W. Smith K.D. Durham A.C. Brown E.J. Combining ATR suppression with oncogenic Ras synergistically increases genomic instability, causing synthetic lethality or tumorigenesis in a dosage-dependent manner.Cancer Res. 2010; 70: 9693-9702Crossref PubMed Scopus (170) Google Scholar, Murga et al., 2011Murga M. Campaner S. Lopez-Contreras A.J. Toledo L.I. Soria R. Montaña M.F. D’Artista L. Schleker T. Guerra C. Garcia E. et al.Exploiting oncogene-induced replicative stress for the selective killing of Myc-driven tumors.Nat. Struct. Mol. Biol. 2011; 18: 1331-1335Crossref PubMed Scopus (289) Google Scholar, Schoppy et al., 2012Schoppy D.W. Ragland R.L. Gilad O. Shastri N. Peters A.A. Murga M. Fernandez-Capetillo O. Diehl J.A. Brown E.J. Oncogenic stress sensitizes murine cancers to hypomorphic suppression of ATR.J. Clin. Invest. 2012; 122: 241-252Crossref PubMed Scopus (139) Google Scholar), leading to the hypothesis that ATR is important for countering the replication stress in cancer cells. Nevertheless, how replication stress can be measured in normal and cancer cells remains elusive. Because multiple ATRi and Chk1i are being tested in clinical trials for cancer therapy (Foote et al., 2013Foote K.M. Blades K. Cronin A. Fillery S. Guichard S.S. Hassall L. Hickson I. Jacq X. Jewsbury P.J. McGuire T.M. et al.Discovery of 4-4-[(3R)-3-Methylmorpholin-4-yl]-6-[1-(methylsulfonyl)cyclopropyl]pyrimidin-2-yl-1H-indole (AZ20): a potent and selective inhibitor of ATR protein kinase with monotherapy in vivo antitumor activity.J. Med. Chem. 2013; 56: 2125-2138Crossref PubMed Scopus (173) Google Scholar, Jossé et al., 2014Jossé R. Martin S.E. Guha R. Ormanoglu P. Pfister T.D. Reaper P.M. Barnes C.S. Jones J. Charlton P. Pollard J.R. et al.ATR inhibitors VE-821 and VX-970 sensitize cancer cells to topoisomerase i inhibitors by disabling DNA replication initiation and fork elongation responses.Cancer Res. 2014; 74: 6968-6979Crossref PubMed Scopus (119) Google Scholar, Karp et al., 2012Karp J.E. Thomas B.M. Greer J.M. Sorge C. Gore S.D. Pratz K.W. Smith B.D. Flatten K.S. Peterson K. Schneider P. et al.Phase I and pharmacologic trial of cytosine arabinoside with the selective checkpoint 1 inhibitor Sch 900776 in refractory acute leukemias.Clin. Cancer Res. 2012; 18: 6723-6731Crossref PubMed Scopus (92) Google Scholar, Ma et al., 2013Ma C.X. Ellis M.J. Petroni G.R. Guo Z. Cai S.R. Ryan C.E. Craig Lockhart A. Naughton M.J. Pluard T.J. Brenin C.M. et al.A phase II study of UCN-01 in combination with irinotecan in patients with metastatic triple negative breast cancer.Breast Cancer Res. Treat. 2013; 137: 483-492Crossref PubMed Scopus (85) Google Scholar, Sausville et al., 2014Sausville E. Lorusso P. Carducci M. Carter J. Quinn M.F. Malburg L. Azad N. Cosgrove D. Knight R. Barker P. et al.Phase I dose-escalation study of AZD7762, a checkpoint kinase inhibitor, in combination with gemcitabine in US patients with advanced solid tumors.Cancer Chemother. Pharmacol. 2014; 73: 539-549Crossref PubMed Scopus (124) Google Scholar, Seto et al., 2013Seto T. Esaki T. Hirai F. Arita S. Nosaki K. Makiyama A. Kometani T. Fujimoto C. Hamatake M. Takeoka H. et al.Phase I, dose-escalation study of AZD7762 alone and in combination with gemcitabine in Japanese patients with advanced solid tumours.Cancer Chemother. Pharmacol. 2013; 72: 619-627Crossref PubMed Scopus (75) Google Scholar), understanding the mechanisms of action and unique properties of these inhibitors may help to guide their applications in clinical settings. In this study, we used multiple inhibitors and small interfering RNAs (siRNAs) to interrogate the functions of ATR and Chk1 during S phase. Unexpectedly, we found that acute inactivation of ATR in S-phase cells led to two distinct outcomes. Upon ATRi treatment, a fraction of S-phase cells accumulated high levels of single-stranded DNA (ssDNA) and underwent replication catastrophe. In contrast, other S-phase cells initially acquired moderate levels of ssDNA, but subsequently recovered from the ATRi shock through a Chk1-mediated mechanism. The critical role of ATR in suppressing replication catastrophe was traced to its functions in promoting ribonucleotide reductase M2 (RRM2) accumulation and limiting replication origin firing in early S phase. In the ATRi-treated cells escaping from replication catastrophe, ATRi triggered a DNA-PK and Chk1-mediated backup pathway to suppress origin firing. Importantly, the Chk1-mediated backup pathway in ATRi-treated cells creates a threshold of tolerable replication stress, allowing ATRi to selectively kill cells under high replication stress. In contrast to ATRi, Chk1i disrupted the backup pathway and induced cell death even when replication stress was moderate. Notably, the levels of ATRi-induced ssDNA correlated with ATRi-induced cell death in a panel of cell lines, suggesting that ATRi-induced ssDNA is a quantitative indicator of replication stress that could be used to predict the ATRi sensitivity of cancer cells. To investigate how ATR functions in cycling cells, we acutely inactivated ATR in U2OS cells with the ATRi VE-821 and followed the effects over time (Figures S1A and S1B; Reaper et al., 2011Reaper P.M. Griffiths M.R. Long J.M. Charrier J.D. Maccormick S. Charlton P.A. Golec J.M. Pollard J.R. Selective killing of ATM- or p53-deficient cancer cells through inhibition of ATR.Nat. Chem. Biol. 2011; 7: 428-430Crossref PubMed Scopus (456) Google Scholar). To visualize ssDNA, DNA was labeled with BrdU and analyzed by native BrdU staining. An increase of ssDNA was detected in S-phase cells 2 hr after ATRi treatment (Figures 1A–1C and S1C). At 8 hr after ATRi treatment, a fraction (∼5%) of S-phase cells displayed very high levels of ssDNA and became strongly positive for γH2AX and TUNEL staining (Figures 1A–1C, S1D, and S1E), indicating that they were undergoing replication catastrophe (Toledo et al., 2013Toledo L.I. Altmeyer M. Rask M.B. Lukas C. Larsen D.H. Povlsen L.K. Bekker-Jensen S. Mailand N. Bartek J. Lukas J. ATR prohibits replication catastrophe by preventing global exhaustion of RPA.Cell. 2013; 155: 1088-1103Abstract Full Text Full Text PDF PubMed Scopus (538) Google Scholar). Surprisingly, however, the majority of ATRi-treated cells displayed less ssDNA at 8 hr than at 2 hr (Figures 1A–1C). This reduction in ssDNA was not due to loss of S-phase cells (Figure S1F). Consistent with the induction of ssDNA at 2 hr, increased amounts of RPA were detected on chromatin by fractionation and immunostaining (Figures 1D and S1G). Subsequently, the levels of RPA on chromatin gradually declined. A similar decline of chromatin-bound RPA also was observed in cells treated with two other ATRis, AZ20 and EPT-46464 (Figure 1E; Foote et al., 2013Foote K.M. Blades K. Cronin A. Fillery S. Guichard S.S. Hassall L. Hickson I. Jacq X. Jewsbury P.J. McGuire T.M. et al.Discovery of 4-4-[(3R)-3-Methylmorpholin-4-yl]-6-[1-(methylsulfonyl)cyclopropyl]pyrimidin-2-yl-1H-indole (AZ20): a potent and selective inhibitor of ATR protein kinase with monotherapy in vivo antitumor activity.J. Med. Chem. 2013; 56: 2125-2138Crossref PubMed Scopus (173) Google Scholar, Toledo et al., 2011Toledo L.I. Murga M. Zur R. Soria R. Rodriguez A. Martinez S. Oyarzabal J. Pastor J. Bischoff J.R. Fernandez-Capetillo O. A cell-based screen identifies ATR inhibitors with synthetic lethal properties for cancer-associated mutations.Nat. Struct. Mol. Biol. 2011; 18: 721-727Crossref PubMed Scopus (374) Google Scholar). Despite the overall decline of chromatin-bound RPA, phosphorylated RPA32 and γH2AX gradually accumulated on chromatin in a fraction of cells after 2 hr (Figures 1A–1D and S1H). Thus, while a subpopulation of ATRi-treated cells acquired high levels of ssDNA and DNA damage, a distinct cell subpopulation gradually recovered. These results raise an important question as to how the distinct effects of ATRi are manifested in S-phase cells. The distinct effects of ATRi on S-phase cells prompted us to investigate whether a fraction of replicating cells are particularly vulnerable to ATR inactivation. Consistent with the induction of ssDNA by ATRi, the staining of chromatin-bound RPA in individual cells gradually increased during the first 2 hr (Figure 2A). When cells were sorted according to EdU incorporation, DNA content, and RPA staining, it was evident that the chromatin binding of RPA occurred most efficiently in a fraction of cells in early-to-mid S phase (Figure 2B). To test more directly if early or mid S-phase cells are most vulnerable to ATR inactivation, we treated synchronously growing T98G cells with ATRi in different stages of the cell cycle (Figures 2C and S2). T98G cells were synchronized in G0 by serum starvation and then released into the cell cycle. Even in the absence of ATRi, low levels of ssDNA were detected in replicating cells (Figure 2D). Interestingly, the basal levels of ssDNA in replicating cells peaked in early S phase, suggesting that cells in this cell-cycle window are facing relatively high levels of intrinsic replication stress. Furthermore, ATRi induced higher levels of ssDNA in early S-phase cells than in mid or late S-phase cells (Figure 2D), suggesting that ATR is particularly important for the suppression of ssDNA in early S phase. To understand why early S-phase cells are vulnerable to ATR inactivation, we tested if any DNA replication factor is limiting during this period. In the presence of HU, ATRi induces excessive firing of replication origins and a massive increase of stalled replication forks, which ultimately leads to exhaustion of RPA and replication catastrophe (Toledo et al., 2013Toledo L.I. Altmeyer M. Rask M.B. Lukas C. Larsen D.H. Povlsen L.K. Bekker-Jensen S. Mailand N. Bartek J. Lukas J. ATR prohibits replication catastrophe by preventing global exhaustion of RPA.Cell. 2013; 155: 1088-1103Abstract Full Text Full Text PDF PubMed Scopus (538) Google Scholar). Even in the absence of HU, ATRi induced a surge of origin firing in 2 hr (Figure S3A). To test if RPA is limiting in early S phase, we monitored the levels of all three RPA subunits in synchronously growing T98G cells (Figure 3A). The levels of RPA70, RPA32, and RPA14 were constant throughout the cell cycle and not affected by ATRi, ruling out RPA as the limiting factor in early S phase. In contrast to RPA, RRM2, a cell-cycle-regulated subunit of the ribonucleotide reductase, gradually accumulated in early S phase (Figure 3A; Chabes et al., 2003Chabes A.L. Pfleger C.M. Kirschner M.W. Thelander L. Mouse ribonucleotide reductase R2 protein: a new target for anaphase-promoting complex-Cdh1-mediated proteolysis.Proc. Natl. Acad. Sci. USA. 2003; 100: 3925-3929Crossref PubMed Scopus (135) Google Scholar, D’Angiolella et al., 2012D’Angiolella V. Donato V. Forrester F.M. Jeong Y.T. Pellacani C. Kudo Y. Saraf A. Florens L. Washburn M.P. Pagano M. Cyclin F-mediated degradation of ribonucleotide reductase M2 controls genome integrity and DNA repair.Cell. 2012; 149: 1023-1034Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar). Notably, ATRi attenuated the accumulation of RRM2 in S phase (Figure 3A). Even in asynchronous U2OS cells, ATRi and Chk1i (MK-8776) reduced the levels of RRM2 (Figures 3B and S3B), suggesting that the ATR-Chk1 pathway promotes RRM2 accumulation in cycling cells. Surprisingly, although RRM2 is an unstable protein, its degradation was not enhanced by ATRi in cells treated with cycloheximide (CHX) (Figure 3C). Knockdown of Cyclin F, the F-box protein required for RRM2 ubiquitylation in G2 (D’Angiolella et al., 2012D’Angiolella V. Donato V. Forrester F.M. Jeong Y.T. Pellacani C. Kudo Y. Saraf A. Florens L. Washburn M.P. Pagano M. Cyclin F-mediated degradation of ribonucleotide reductase M2 controls genome integrity and DNA repair.Cell. 2012; 149: 1023-1034Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar), did not suppress the reduction of RRM2 in ATRi-treated cells (Figure S3C). In contrast to RRM2, E2F1, the transcription activator of the RRM2 gene (DeGregori et al., 1995DeGregori J. Kowalik T. Nevins J.R. Cellular targets for activation by the E2F1 transcription factor include DNA synthesis- and G1/S-regulatory genes.Mol. Cell. Biol. 1995; 15: 4215-4224Crossref PubMed Scopus (837) Google Scholar, Zhang et al., 2009Zhang Y.W. Jones T.L. Martin S.E. Caplen N.J. Pommier Y. Implication of checkpoint kinase-dependent up-regulation of ribonucleotide reductase R2 in DNA damage response.J. Biol. Chem. 2009; 284: 18085-18095Crossref PubMed Scopus (102) Google Scholar), was increasingly degraded in ATRi-treated cells in the presence of CHX (Figure 3C). Similar to RRM2, E2F1 was reduced in ATRi- and Chk1i-treated cells (Figures 3B and S3B). Concomitant with the reduction of E2F1, RRM2 mRNA levels declined (Figure S3D). Importantly, overexpression of E2F1 completely suppressed the reduction of RRM2 in ATRi-treated cells (Figure 3D), suggesting that E2F1 degradation is responsible for the reduction of RRM2 by ATRi. Together, these results show that the ATR-Chk1 pathway promotes RRM2 accumulation by stabilizing E2F1. The ATRi-induced reduction in E2F1 and RRM2 was suppressed by the CDK inhibitor roscovitine, the proteasome inhibitor MG132, and the Nedd8-activating enzyme inhibitor MLN4924 (Figures 3E and 3F; Soucy et al., 2009Soucy T.A. Smith P.G. Milhollen M.A. Berger A.J. Gavin J.M. Adhikari S. Brownell J.E. Burke K.E. Cardin D.P. Critchley S. et al.An inhibitor of NEDD8-activating enzyme as a new approach to treat cancer.Nature. 2009; 458: 732-736Crossref PubMed Scopus (1351) Google Scholar). In contrast, Wee1 inhibitor, which activates CDK1/2 (Beck et al., 2012Beck H. Nähse-Kumpf V. Larsen M.S. O’Hanlon K.A. Patzke S. Holmberg C. Mejlvang J. Groth A. Nielsen O. Syljuåsen R.G. Sørensen C.S. Cyclin-dependent kinase suppression by WEE1 kinase protects the genome through control of replication initiation and nucleotide consumption.Mol. Cell. Biol. 2012; 32: 4226-4236Crossref PubMed Scopus (196) Google Scholar, Hughes et al., 2013Hughes B.T. Sidorova J. Swanger J. Monnat Jr., R.J. Clurman B.E. Essential role for Cdk2 inhibitory phosphorylation during replication stress revealed by a human Cdk2 knockin mutation.Proc. Natl. Acad. Sci. USA. 2013; 110: 8954-8959Crossref PubMed Scopus (41) Google Scholar), drastically reduced E2F1 and RRM2 levels even in the absence of ATRi (Figure 3E). These results suggest that the ATR-Chk1 pathway promotes RRM2 accumulation by antagonizing a CDK1/2, Cullin-RING ubiquitin ligase and proteasome-mediated mechanism that degrades E2F1 (see Figure 3H). In ATRi-treated cells, roscovitine not only elevated RRM2 levels but also reduced the induction of γH2AX (Figure 3E). Furthermore, a CDK2-specific inhibitor also reduced γH2AX (Figure S3E). These results suggest that a reduction in CDK2 activity may suppress ATRi-induced DNA damage by increasing RRM2 levels. Indeed, expression of RRM2 significantly reduced the γH2AX induced by ATRi or Chk1i (Figures 3G and S3F). In addition to its effects on RRM2, roscovitine decreased origin firing in ATRi-treated cells (Figure S3A). To test if suppression of origin firing reduces ATRi-induced DNA damage, we used siRNA to knock down CDC7, a key regulator of replication initiation (Labib, 2010Labib K. How do Cdc7 and cyclin-dependent kinases trigger the initiation of chromosome replication in eukaryotic cells?.Genes Dev. 2010; 24: 1208-1219Crossref PubMed Scopus (277) Google Scholar). Knockdown of CDC7 indeed reduced ATRi-induced γH2AX (Figure S3G). Thus, ATR, through restricting CDK2 activity, promotes RRM2 accumulation and limits origin firing in S phase (Figure 3H). Both of these effects of ATR contribute to the suppression of DNA damage in replicating cells (Figure 3H). How ATR is activated during unperturbed S phase and how E2F1 is suppressed by CDK2 and Cullin ligases remain to be investigated. The transient accumulation of ssDNA in S-phase cells may trigger limited ATR activation, thereby coordinating RRM2 accumulation and origin firing. Interestingly, the budding yeast ATR homolog Mec1 is required for priming the Mcm2-7 helicase for phosphorylation by Cdc7 (Randell et al., 2010Randell J.C. Fan A. Chan C. Francis L.I. Heller R.C. Galani K. Bell S.P. Mec1 is one of multiple kinases that prime the Mcm2-7 helicase for phosphorylation by Cdc7.Mol. Cell. 2010; 40: 353-363Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). The limited ATR activation during S phase may promote origin firing but also restrict it to a tolerable level, preventing ssDNA from accumulating to a high level that triggers replication catastrophe (see Figure 7A). The correlations of high ssDNA with replication catastrophe and moderate ssDNA with recovery suggest that the fate of ATRi-treated cells may be dictated by a threshold of ssDNA (see Figure 7A). To investigate the underlying mechanism of recovery, we first tested if ATRi gradually looses its potency during this proce" @default.
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• W1855851918 date "2015-09-01" @default.
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• W1855851918 title "Distinct but Concerted Roles of ATR, DNA-PK, and Chk1 in Countering Replication Stress during S Phase" @default.
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• W1855851918 doi "https://doi.org/10.1016/j.molcel.2015.07.029" @default.
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