Dbf4 Is Direct Downstream Target of Ataxia Telangiectasia Mutated (ATM) and Ataxia Telangiectasia and Rad3-related (ATR) Protein to Regulate Intra-S-phase Checkpoint

Abstract

Dbf4/Cdc7 (Dbf4-dependent kinase (DDK)) is activated at the onset of S-phase, and its kinase activity is required for DNA replication initiation from each origin. We showed that DDK is an important target for the S-phase checkpoint in mammalian cells to suppress replication initiation and to protect replication forks. We demonstrated that ataxia telangiectasia mutated (ATM) and ataxia telangiectasia and Rad3-related (ATR) proteins directly phosphorylate Dbf4 in response to ionizing radiation and replication stress. We identified novel ATM/ATR phosphorylation sites on Dbf4 and showed that ATM/ATR-mediated phosphorylation of Dbf4 is critical for the intra-S-phase checkpoint to inhibit DNA replication. The kinase activity of DDK, which is not suppressed upon DNA damage, is required for fork protection under replication stress. We further demonstrated that ATM/ATR-mediated phosphorylation of Dbf4 is important for preventing DNA rereplication upon loss of replication licensing through the activation of the S-phase checkpoint. These studies indicate that DDK is a direct substrate of ATM and ATR to mediate the intra-S-phase checkpoint in mammalian cells. Dbf4/Cdc7 (Dbf4-dependent kinase (DDK)) is activated at the onset of S-phase, and its kinase activity is required for DNA replication initiation from each origin. We showed that DDK is an important target for the S-phase checkpoint in mammalian cells to suppress replication initiation and to protect replication forks. We demonstrated that ataxia telangiectasia mutated (ATM) and ataxia telangiectasia and Rad3-related (ATR) proteins directly phosphorylate Dbf4 in response to ionizing radiation and replication stress. We identified novel ATM/ATR phosphorylation sites on Dbf4 and showed that ATM/ATR-mediated phosphorylation of Dbf4 is critical for the intra-S-phase checkpoint to inhibit DNA replication. The kinase activity of DDK, which is not suppressed upon DNA damage, is required for fork protection under replication stress. We further demonstrated that ATM/ATR-mediated phosphorylation of Dbf4 is important for preventing DNA rereplication upon loss of replication licensing through the activation of the S-phase checkpoint. These studies indicate that DDK is a direct substrate of ATM and ATR to mediate the intra-S-phase checkpoint in mammalian cells. IntroductionThe initiation of DNA replication requires a series of coordinated events during the cell cycle (1Kelly T.J. Brown G.W. Regulation of chromosome replication.Annu. Rev. Biochem. 2000; 69: 829-880Crossref PubMed Scopus (333) Google Scholar, 2Bell S.P. Dutta A. DNA replication in eukaryotic cells.Annu. Rev. Biochem. 2002; 71: 333-374Crossref PubMed Scopus (1388) Google Scholar). In late mitosis and G1, MCM2-7 proteins are loaded onto chromatin to form prereplicative complexes. When cells enter S-phase, S-phase cyclin-dependent kinases and Dbf4/Cdc7 (DDK) 4The abbreviations used are: DDKDbf4-dependent kinaseATMataxia telangiectasia mutatedATRataxia telangiectasia and Rad3-relatedDSBdouble-stranded breakRPAreplication protein AHUhydroxyureaGygraysAdadenovirus. are required for activating prereplicative complexes by recruiting replication initiation proteins, such as Cdc45 and GINS, to origins (3Lei M. Tye B.K. Initiating DNA synthesis. From recruiting to activating the MCM complex.J. Cell Sci. 2001; 114: 1447-1454Crossref PubMed Google Scholar, 4Labib 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 (272) Google Scholar).Cdc7 is a serine/threonine kinase, conserved from yeast to humans (5Johnston L.H. Masai H. Sugino A. A Cdc7p-Dbf4p protein kinase activity is conserved from yeast to humans.Prog. Cell Cycle Res. 2000; 4: 61-69Crossref PubMed Scopus (16) Google Scholar). It is activated by binding to its regulatory protein Dbf4 (6Jackson A.L. Pahl P.M. Harrison K. Rosamond J. Sclafani R.A. Cell cycle regulation of the yeast Cdc7 protein kinase by association with the Dbf4 protein.Mol. Cell Biol. 1993; 13: 2899-2908Crossref PubMed Scopus (212) Google Scholar, 7Jiang W. McDonald D. Hope T.J. Hunter T. Mammalian Cdc7-Dbf4 protein kinase complex is essential for initiation of DNA replication.EMBO J. 1999; 18: 5703-5713Crossref PubMed Scopus (169) Google Scholar). Cdc7 protein levels appear constant during the cell cycle, whereas its kinase activity peaks at the G1/S transition due to the cell cycle-regulated oscillations in Dbf4 protein levels (6Jackson A.L. Pahl P.M. Harrison K. Rosamond J. Sclafani R.A. Cell cycle regulation of the yeast Cdc7 protein kinase by association with the Dbf4 protein.Mol. Cell Biol. 1993; 13: 2899-2908Crossref PubMed Scopus (212) Google Scholar, 8Yoon H.J. Loo S. Campbell J.L. Regulation of Saccharomyces cerevisiae CDC7 function during the cell cycle.Mol. Biol. Cell. 1993; 4: 195-208Crossref PubMed Scopus (64) Google Scholar, 9Cheng L. Collyer T. Hardy C.F. Cell cycle regulation of DNA replication initiator factor Dbf4p.Mol. Cell Biol. 1999; 19: 4270-4278Crossref PubMed Scopus (82) Google Scholar, 10Pasero P. Duncker B.P. Schwob E. Gasser S.M. A role for the Cdc7 kinase regulatory subunit Dbf4p in the formation of initiation-competent origins of replication.Genes Dev. 1999; 13: 2159-2176Crossref PubMed Scopus (100) Google Scholar). DDK is required for the initiation of DNA replication from each individual origin, including both early and late firing origins (11Donaldson A.D. Fangman W.L. Brewer B.J. Cdc7 is required throughout the yeast S phase to activate replication origins.Genes Dev. 1998; 12: 491-501Crossref PubMed Scopus (182) Google Scholar, 12Bousset K. Diffley J.F. The Cdc7 protein kinase is required for origin firing during S phase.Genes Dev. 1998; 12: 480-490Crossref PubMed Scopus (238) Google Scholar). The MCM2-7 complex is an important target of DDK for DNA replication initiation (13Jares P. Donaldson A. Blow J.J. The Cdc7/Dbf4 protein kinase. Target of the S phase checkpoint?.EMBO Rep. 2000; 1: 319-322Crossref PubMed Scopus (74) Google Scholar).The S-phase checkpoint is activated in response to replication stress or DNA lesions that occur in S-phase and is critical for the maintenance of genome stability (14Bartek J. Lukas C. Lukas J. Checking on DNA damage in S phase.Nat. Rev. Mol. Cell Biol. 2004; 5: 792-804Crossref PubMed Scopus (588) Google Scholar, 15Aguilera A. Gómez-González B. Genome instability. A mechanistic view of its causes and consequences.Nat. Rev. Genet. 2008; 9: 204-217Crossref PubMed Scopus (557) Google Scholar). The S-phase checkpoint inhibits late replication origin firing to slow down DNA replication in response to DNA damage in S-phase. It also stabilizes and protects stalled replication forks upon replication stress and facilitates replication restart during recovery from fork stalling or damage. Ataxia telangiectasia mutated (ATM) and ataxia telangiectasia and Rad3-related protein (ATR) are two major phosphatidylinositol 3-like protein kinases that are critical for detecting aberrant DNA structures (16Abraham R.T. Cell cycle checkpoint signaling through the ATM and ATR kinases.Genes Dev. 2001; 15: 2177-2196Crossref PubMed Scopus (1652) Google Scholar). ATM in collaboration with the Mre11/Rad50/Nbs1 complex participates in the detection of DNA double-stranded breaks (DSBs), leading to the activation of the Chk2 pathway. ATR with its partner ATR-interacting protein detects DNA lesions by recognizing replication protein A (RPA)-coated single-stranded DNA (ssDNA) and activates the Chk1 pathway (17Cimprich K.A. Cortez D. ATR. An essential regulator of genome integrity.Nat. Rev. Mol. Cell Biol. 2008; 9: 616-627Crossref PubMed Scopus (1296) Google Scholar, 18Lee J.H. Paull T.T. Activation and regulation of ATM kinase activity in response to DNA double-strand breaks.Oncogene. 2007; 26: 7741-7748Crossref PubMed Scopus (407) Google Scholar).In yeast, substantial evidence suggests that DDK is a direct target of the S-phase checkpoint. Dbf4/Dfp1 (Dfp1:Dbf4 ortholog in Schizosaccharomyces pombe) undergoes Rad53/Cds1 (orthologs of Chk2 in yeast)-dependent phosphorylation upon HU treatment (19Brown G.W. Kelly T.J. Cell cycle regulation of Dfp1, an activator of the Hsk1 protein kinase.Proc. Natl. Sci. 1999; PubMed Scopus Google Scholar, Brown G.W. Schizosaccharomyces is a target required for genome Cell Biol. 2000; PubMed Scopus Google Scholar, M. B. Cdc7p-Dbf4p kinase to chromatin during S phase and is by both the and the checkpoint J. 1999; 18: PubMed Scopus Google Scholar). The chromatin binding of Dbf4 is HU treatment in a P. Duncker B.P. Schwob E. Gasser S.M. A role for the Cdc7 kinase regulatory subunit Dbf4p in the formation of initiation-competent origins of replication.Genes Dev. 1999; 13: 2159-2176Crossref PubMed Scopus (100) Google and phosphorylation of Dbf4 the kinase activity of DDK M. B. Cdc7p-Dbf4p kinase to chromatin during S phase and is by both the and the checkpoint J. 1999; 18: PubMed Scopus Google Scholar). studies further demonstrated that phosphorylation of Dbf4 directly inhibits origin firing to mediate the intra-S-phase checkpoint in yeast J. J. phosphorylation of is important to late origin 2010; PubMed Scopus Google Scholar, P. Diffley J.F. of DNA replication initiation by and Dbf4 2010; PubMed Scopus Google Scholar). of Dbf4 and Cdc7 and that DDK a critical role in fork and recovery from replication fork in yeast Brown G.W. Schizosaccharomyces is a target required for genome Cell Biol. 2000; PubMed Scopus Google Scholar, S. K. E. P. Masai H. the Cdc7-Dbf4 kinase in Schizosaccharomyces with a of the replication fork protection complex.J. Biol. PubMed Scopus Google Scholar, T. K. E. H. T. K. Masai H. A yeast a regulatory subunit for Hsk1 kinase, essential in S-phase initiation as as in S-phase checkpoint and recovery from DNA Cell Biol. 1999; 19: PubMed Scopus Google Scholar).In the that a DNA activates the S-phase checkpoint and inhibits the kinase activity of DDK D. K.A. M. J. An and DNA damage checkpoint that inhibits initiation of DNA Cell. PubMed Scopus Google Scholar). of of DDK activity in cells with J. M. to the and an phase Cell. 2004; 5: PubMed Scopus Google Scholar). studies in both and mammalian cells demonstrated that DDK during the S-phase checkpoint response T. E. W. The role of kinase Cdc7 in DNA damage checkpoint Cell. 2008; PubMed Scopus Google Scholar, K. A. M. Cdc7-Dbf4 and the S checkpoint response to Biol. 2007; PubMed Scopus Google Scholar, P. T. C. The S-phase checkpoint initiation factor Cdc45 a Biol. PubMed Scopus Google Scholar, P. Hunter T. ATM and ATR in a and DNA damage Cell Biol. 26: PubMed Scopus Google Scholar, T. M. complex is required for the initiation of DNA Biol. PubMed Scopus Google Scholar, P. D. A. S. C. C. Cdc7 is an kinase in cells replication Biol. 2007; PubMed Scopus Google Scholar). In a to a role important that of Dbf4 in the S-phase checkpoint T. M. complex is required for the initiation of DNA Biol. PubMed Scopus Google Scholar, is a protein kinase required for the initiation of DNA replication.Genes Dev. 19: PubMed Scopus Google Scholar). These studies a as to DDK is a critical S-phase checkpoint target to inhibit replication initiation its kinase activity is not DNA damage in demonstrated that Dbf4 is a direct target of ATM and ATR the S-phase checkpoint is We identified ATM/ATR phosphorylation sites on Dbf4 and showed that phosphorylation of Dbf4 is important for replication initiation to mediate the intra-S-phase checkpoint through a of DDK kinase also that the kinase activity of DDK is required for replication forks upon replication stress. Dbf4 to mediate the S-phase checkpoint in mammalian cells. is a direct target of the S-phase checkpoint to inhibit DNA DDK to protect stalled replication forks. with its role in replication initiation upon the activation of S-phase also demonstrated that ATM/ATR-mediated phosphorylation of Dbf4 is important for the of DNA rereplication the licensing is essential in DNA replication at origins during each cell cycle (3Lei M. Tye B.K. Initiating DNA synthesis. From recruiting to activating the MCM complex.J. Cell Sci. 2001; 114: 1447-1454Crossref PubMed Google Scholar, H. K. Cdc7 kinase A regulator in the initiation of DNA Cell 2002; PubMed Scopus Google Scholar). in yeast substantial evidence that DDK also participates in the S-phase checkpoint and fork protection upon DNA damage and replication stress (13Jares P. Donaldson A. Blow J.J. The Cdc7/Dbf4 protein kinase. Target of the S phase checkpoint?.EMBO Rep. 2000; 1: 319-322Crossref PubMed Scopus (74) Google Scholar, B.P. Brown G.W. Cdc7 kinases and checkpoint from two Res. PubMed Scopus Google Scholar). The S-phase checkpoint function is conserved in (14Bartek J. Lukas C. Lukas J. Checking on DNA damage in S phase.Nat. Rev. Mol. Cell Biol. 2004; 5: 792-804Crossref PubMed Scopus (588) Google is DDK is an important target for the intra-S-phase checkpoint to suppress replication initiation in mammalian cells. The function of DDK in fork protection in mammalian cells also that in and in mammalian DDK an important role in replication during the S-phase checkpoint recovery T. E. W. The role of kinase Cdc7 in DNA damage checkpoint Cell. 2008; PubMed Scopus Google Scholar). In demonstrated that DDK also of the as a direct target of ATM and ATR to mediate the intra-S-phase checkpoint We that Dbf4 is upon DNA damage or replication and events on ATM ATR ATM and ATR directly phosphorylate Dbf4 in and the ATM/ATR conserved phosphorylation sites of Dbf4 in the that Dbf4 is directly by ATM ATR upon the activation of is to yeast, is directly by the S-phase checkpoint through phosphorylation cells are with HU (19Brown G.W. Kelly T.J. Cell cycle regulation of Dfp1, an activator of the Hsk1 protein kinase.Proc. Natl. Sci. 1999; PubMed Scopus Google Scholar, Brown G.W. Schizosaccharomyces is a target required for genome Cell Biol. 2000; PubMed Scopus Google Scholar, M. B. Cdc7p-Dbf4p kinase to chromatin during S phase and is by both the and the checkpoint J. 1999; 18: PubMed Scopus Google Scholar). These studies as in the Dbf4 in mammalian cells is also a target of the intra-S-phase checkpoint to suppress replication initiation of late origins in an S-phase checkpoint that the direct phosphorylation of Dbf4 by in yeast inhibits the kinase activity of DDK, preventing late origins from firing to mediate the S-phase checkpoint M. W. S. A. K. L.H. Sugino A. of the yeast complex from cells. protein kinase activity is by Biol. 2000; PubMed Scopus Google Scholar). as to DDK activity is upon DNA damage D. K.A. M. J. An and DNA damage checkpoint that inhibits initiation of DNA Cell. PubMed Scopus Google Scholar, T. E. W. The role of kinase Cdc7 in DNA damage checkpoint Cell. 2008; PubMed Scopus Google Scholar). We demonstrated by both in and in that in mammalian phosphorylation of Dbf4 of Dbf4 and Cdc7 inhibits the kinase activity of the DDK complex for Dbf4 and These that of the intra-S-phase checkpoint by phosphorylation of Dbf4 is not through the kinase activity of the in S-phase checkpoint pathway is that phosphorylation of the ATM/ATR sites on Dbf4 sites to or to the of replication origins to replication the phosphorylation events the of replication initiation proteins with DDK and DNA replication It is also that DDK upon DNA damage, ATM/ATR-mediated phosphorylation its with of its that are in replication the phosphorylation of is due to the of the of DDK with DDK kinase activity upon DNA damage. It such the the role of DDK to mediate the S-phase checkpoint in mammalian cells is of a to the intra-S-phase checkpoint through the pathway DDK kinase activity important in of DNA damage for the maintenance of genome showed that DDK as as its kinase activity is for fork protection upon replication stress. The chromatin of DDK is replication forks are stalled by HU and of the of Dbf4 or Cdc7 by to under replication and the kinase activity of Cdc7 important for fork is with the that yeast Cdc7 and Dbf4 in cell and intra-S-phase are to HU and and and replication stress Brown G.W. Schizosaccharomyces is a target required for genome Cell Biol. 2000; PubMed Scopus Google Scholar, M. B. Cdc7p-Dbf4p kinase to chromatin during S phase and is by both the and the checkpoint J. 1999; 18: PubMed Scopus Google Scholar, A.D. J. S. Brown G.W. A conserved of Schizosaccharomyces is required for chromosome stability damage during S Cell Biol. 2002; PubMed Scopus Google Scholar). It also that DDK to and the of the fork protection and its kinase activity is required for and maintenance of the of fork protection upon replication fork S. K. E. P. Masai H. the Cdc7-Dbf4 kinase in Schizosaccharomyces with a of the replication fork protection complex.J. Biol. PubMed Scopus Google Scholar, E. S. Schizosaccharomyces and Hsk1 are of a novel S-phase response pathway to Cell. Biol. PubMed Scopus Google Scholar, S. M. M. M. P. Masai H. Hsk1 kinase and Cdc45 replication checkpoint in 2010; 9: PubMed Scopus Google Scholar). in mammalian studies showed that Cdc7 with the factor and that phosphorylation of is for phosphorylation upon replication stress S. Masai H. and of replication from mammalian cells. DNA binding activity and novel protein PubMed Scopus Google Scholar, M. Masai H. Cdc7 kinase phosphorylation in DNA replication 2008; PubMed Scopus Google Scholar). the kinase activity of DDK to checkpoint signaling and DNA studies showed that in yeast, DDK a checkpoint in response to DNA damage, and phosphorylation the of from damage sites K. Masai H. H. DDK checkpoint and its from Cell. 2010; PubMed Scopus Google Scholar). The in phosphorylation are in formation and replication a in DNA the kinase activity of DDK is to important for activating replication initiation during S-phase recovery T. E. W. The role of kinase Cdc7 in DNA damage checkpoint Cell. 2008; PubMed Scopus Google Scholar). of replication stress of DNA in S-phase, of DNA replication requires phosphorylation of critical replication initiation DDK and its kinase activity important for replication replication and cell cycle, which is by the replication licensing loss of the replication licensing DNA rereplication is which to genome instability. We showed that the S-phase checkpoint is activated to suppress DNA rereplication by of the licensing protein E. T. E. P. The S phase checkpoint rereplication in mammalian cells licensing is Cell Biol. 2007; PubMed Scopus Google Scholar). In that Dbf4 is by ATR at the early is and phosphorylation is important for DNA We that DDK is an important target of the S-phase checkpoint to inhibit DNA and the used in response is to that of the intra-S-phase checkpoint to suppress DNA replication initiation Dbf4 is by ATM a role to phosphorylate Dbf4 in response to to mediate the intra-S-phase ATR is important for Dbf4 at the early upon loss of the licensing of DNA rereplication E. T. E. P. The S phase checkpoint rereplication in mammalian cells licensing is Cell Biol. 2007; PubMed Scopus Google Scholar). ATM is activated late to phosphorylate Dbf4 are during rereplication studies that DDK is an important target of the intra-S-phase checkpoint through direct phosphorylation of Dbf4 by also to that DDK is not the critical target for the intra-S-phase checkpoint replication proteins, such as and MCM proteins, are also in the intra-S-phase checkpoint E. T. E. P. The S phase checkpoint rereplication in mammalian cells licensing is Cell Biol. 2007; PubMed Scopus Google Scholar, E. E. is a direct target for ATR to the S-phase Biol. PubMed Scopus Google Scholar, D. maintenance proteins are direct of the ATM and ATR checkpoint Natl. Sci. 2004; PubMed Scopus Google Scholar). It is important to replication initiation proteins are upon checkpoint activation and function to the intra-S-phase checkpoint. studies also that the the function of DDK in the intra-S-phase checkpoint and in the fork protection are phosphorylation of Dbf4 the intra-S-phase checkpoint response through a its kinase the fork protection of DDK requires its kinase DDK its kinase activity for fork DNA and checkpoint recovery in response to replication stress a critical target for replication initiation upon the activation of S-phase checkpoint. studies a mechanistic for further the of the regulation of DDK function to mediate the S-phase checkpoint and to genome stability in S-phase. IntroductionThe initiation of DNA replication requires a series of coordinated events during the cell cycle (1Kelly T.J. Brown G.W. Regulation of chromosome replication.Annu. Rev. Biochem. 2000; 69: 829-880Crossref PubMed Scopus (333) Google Scholar, 2Bell S.P. Dutta A. DNA replication in eukaryotic cells.Annu. Rev. Biochem. 2002; 71: 333-374Crossref PubMed Scopus (1388) Google Scholar). In late mitosis and G1, MCM2-7 proteins are loaded onto chromatin to form prereplicative complexes. When cells enter S-phase, S-phase cyclin-dependent kinases and Dbf4/Cdc7 (DDK) 4The abbreviations used are: DDKDbf4-dependent kinaseATMataxia telangiectasia mutatedATRataxia telangiectasia and Rad3-relatedDSBdouble-stranded breakRPAreplication protein AHUhydroxyureaGygraysAdadenovirus. are required for activating prereplicative complexes by recruiting replication initiation proteins, such as Cdc45 and GINS, to origins (3Lei M. Tye B.K. Initiating DNA synthesis. From recruiting to activating the MCM complex.J. Cell Sci. 2001; 114: 1447-1454Crossref PubMed Google Scholar, 4Labib 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 (272) Google Scholar).Cdc7 is a serine/threonine kinase, conserved from yeast to humans (5Johnston L.H. Masai H. Sugino A. A Cdc7p-Dbf4p protein kinase activity is conserved from yeast to humans.Prog. Cell Cycle Res. 2000; 4: 61-69Crossref PubMed Scopus (16) Google Scholar). It is activated by binding to its regulatory protein Dbf4 (6Jackson A.L. Pahl P.M. Harrison K. Rosamond J. Sclafani R.A. Cell cycle regulation of the yeast Cdc7 protein kinase by association with the Dbf4 protein.Mol. Cell Biol. 1993; 13: 2899-2908Crossref PubMed Scopus (212) Google Scholar, 7Jiang W. McDonald D. Hope T.J. Hunter T. Mammalian Cdc7-Dbf4 protein kinase complex is essential for initiation of DNA replication.EMBO J. 1999; 18: 5703-5713Crossref PubMed Scopus (169) Google Scholar). Cdc7 protein levels appear constant during the cell cycle, whereas its kinase activity peaks at the G1/S transition due to the cell cycle-regulated oscillations in Dbf4 protein levels (6Jackson A.L. Pahl P.M. Harrison K. Rosamond J. Sclafani R.A. Cell cycle regulation of the yeast Cdc7 protein kinase by association with the Dbf4 protein.Mol. Cell Biol. 1993; 13: 2899-2908Crossref PubMed Scopus (212) Google Scholar, 8Yoon H.J. Loo S. Campbell J.L. Regulation of Saccharomyces cerevisiae CDC7 function during the cell cycle.Mol. Biol. Cell. 1993; 4: 195-208Crossref PubMed Scopus (64) Google Scholar, 9Cheng L. Collyer T. Hardy C.F. Cell cycle regulation of DNA replication initiator factor Dbf4p.Mol. Cell Biol. 1999; 19: 4270-4278Crossref PubMed Scopus (82) Google Scholar, 10Pasero P. Duncker B.P. Schwob E. Gasser S.M. A role for the Cdc7 kinase regulatory subunit Dbf4p in the formation of initiation-competent origins of replication.Genes Dev. 1999; 13: 2159-2176Crossref PubMed Scopus (100) Google Scholar). DDK is required for the initiation of DNA replication from each individual origin, including both early and late firing origins (11Donaldson A.D. Fangman W.L. Brewer B.J. Cdc7 is required throughout the yeast S phase to activate replication origins.Genes Dev. 1998; 12: 491-501Crossref PubMed Scopus (182) Google Scholar, 12Bousset K. Diffley J.F. The Cdc7 protein kinase is required for origin firing during S phase.Genes Dev. 1998; 12: 480-490Crossref PubMed Scopus (238) Google Scholar). The MCM2-7 complex is an important target of DDK for DNA replication initiation (13Jares P. Donaldson A. Blow J.J. The Cdc7/Dbf4 protein kinase. Target of the S phase checkpoint?.EMBO Rep. 2000; 1: 319-322Crossref PubMed Scopus (74) Google Scholar).The S-phase checkpoint is activated in response to replication stress or DNA lesions that occur in S-phase and is critical for the maintenance of genome stability (14Bartek J. Lukas C. Lukas J. Checking on DNA damage in S phase.Nat. Rev. Mol. Cell Biol. 2004; 5: 792-804Crossref PubMed Scopus (588) Google Scholar, 15Aguilera A. Gómez-González B. Genome instability. A mechanistic view of its causes and consequences.Nat. Rev. Genet. 2008; 9: 204-217Crossref PubMed Scopus (557) Google Scholar). The S-phase checkpoint inhibits late replication origin firing to slow down DNA replication in response to DNA damage in S-phase. It also stabilizes and protects stalled replication forks upon replication stress and facilitates replication restart during recovery from fork stalling or damage. Ataxia telangiectasia mutated (ATM) and ataxia telangiectasia and Rad3-related protein (ATR) are two major phosphatidylinositol 3-like protein kinases that are critical for detecting aberrant DNA structures (16Abraham R.T. Cell cycle checkpoint signaling through the ATM and ATR kinases.Genes Dev. 2001; 15: 2177-2196Crossref PubMed Scopus (1652) Google Scholar). ATM in collaboration with the Mre11/Rad50/Nbs1 complex participates in the detection of DNA double-stranded breaks (DSBs), leading to the activation of the Chk2 pathway. ATR with its partner ATR-interacting protein detects DNA lesions by recognizing replication protein A (RPA)-coated single-stranded DNA (ssDNA) and activates the Chk1 pathway (17Cimprich K.A. Cortez D. ATR. An essential regulator of genome integrity.Nat. Rev. Mol. Cell Biol. 2008; 9: 616-627Crossref PubMed Scopus (1296) Google Scholar, 18Lee J.H. Paull T.T. Activation and regulation of ATM kinase activity in response to DNA double-strand breaks.Oncogene. 2007; 26: 7741-7748Crossref PubMed Scopus (407) Google Scholar).In yeast, substantial evidence suggests that DDK is a direct target of the S-phase checkpoint. Dbf4/Dfp1 (Dfp1:Dbf4 ortholog in Schizosaccharomyces pombe) undergoes Rad53/Cds1 (orthologs of Chk2 in yeast)-dependent phosphorylation upon HU treatment (19Brown G.W. Kelly T.J. Cell cycle regulation of Dfp1, an activator of the Hsk1 protein kinase.Proc. Natl. Sci. 1999; PubMed Scopus Google Scholar, Brown G.W. Schizosaccharomyces is a target required for genome Cell Biol. 2000; PubMed Scopus Google Scholar, M. B. Cdc7p-Dbf4p kinase to chromatin during S phase and is by both the and the checkpoint J. 1999; 18: PubMed Scopus Google Scholar). The chromatin binding of Dbf4 is HU treatment in a P. Duncker B.P. Schwob E. Gasser S.M. A role for the Cdc7 kinase regulatory subunit Dbf4p in the formation of initiation-competent origins of replication.Genes Dev. 1999; 13: 2159-2176Crossref PubMed Scopus (100) Google and phosphorylation of Dbf4 the kinase activity of DDK M. B. Cdc7p-Dbf4p kinase to chromatin during S phase and is by both the and the checkpoint J. 1999; 18: PubMed Scopus Google Scholar). studies further demonstrated that phosphorylation of Dbf4 directly inhibits origin firing to mediate the intra-S-phase checkpoint in yeast J. J. phosphorylation of is important to late origin 2010; PubMed Scopus Google Scholar, P. Diffley J.F. of DNA replication initiation by and Dbf4 2010; PubMed Scopus Google Scholar). of Dbf4 and Cdc7 and that DDK a critical role in fork and recovery from replication fork in yeast Brown G.W. Schizosaccharomyces is a target required for genome Cell Biol. 2000; PubMed Scopus Google Scholar, S. K. E. P. Masai H. the Cdc7-Dbf4 kinase in Schizosaccharomyces with a of the replication fork protection complex.J. Biol. PubMed Scopus Google Scholar, T. K. E. H. T. K. Masai H. A yeast a regulatory subunit for Hsk1 kinase, essential in S-phase initiation as as in S-phase checkpoint and recovery from DNA Cell Biol. 1999; 19: PubMed Scopus Google Scholar).In the that a DNA activates the S-phase checkpoint and inhibits the kinase activity of DDK D. K.A. M. J. An and DNA damage checkpoint that inhibits initiation of DNA Cell. PubMed Scopus Google Scholar). of of DDK activity in cells with J. M. to the and an phase Cell. 2004; 5: PubMed Scopus Google Scholar). studies in both and mammalian cells demonstrated that DDK during the S-phase checkpoint response T. E. W. The role of kinase Cdc7 in DNA damage checkpoint Cell. 2008; PubMed Scopus Google Scholar, K. A. M. Cdc7-Dbf4 and the S checkpoint response to Biol. 2007; PubMed Scopus Google Scholar, P. T. C. The S-phase checkpoint initiation factor Cdc45 a Biol. PubMed Scopus Google Scholar, P. Hunter T. ATM and ATR in a and DNA damage Cell Biol. 26: PubMed Scopus Google Scholar, T. M. complex is required for the initiation of DNA Biol. PubMed Scopus Google Scholar, P. D. A. S. C. C. Cdc7 is an kinase in cells replication Biol. 2007; PubMed Scopus Google Scholar). In a to a role important that of Dbf4 in the S-phase checkpoint T. M. complex is required for the initiation of DNA Biol. PubMed Scopus Google Scholar, is a protein kinase required for the initiation of DNA replication.Genes Dev. 19: PubMed Scopus Google Scholar). These studies a as to DDK is a critical S-phase checkpoint target to inhibit replication initiation its kinase activity is not DNA damage in demonstrated that Dbf4 is a direct target of ATM and ATR the S-phase checkpoint is We identified ATM/ATR phosphorylation sites on Dbf4 and showed that phosphorylation of Dbf4 is important for replication initiation to mediate the intra-S-phase checkpoint through a of DDK kinase also that the kinase activity of DDK is required for replication forks upon replication stress. Dbf4 to mediate the S-phase checkpoint in mammalian cells. is a direct target of the S-phase checkpoint to inhibit DNA DDK to protect stalled replication forks. with its role in replication initiation upon the activation of S-phase also demonstrated that ATM/ATR-mediated phosphorylation of Dbf4 is important for the of DNA rereplication the licensing is