Mcm2 Is a Direct Substrate of ATM and ATR during DNA Damage and DNA Replication Checkpoint Responses

Yoo, Hae Young; Shevchenko, Anna; Dunphy, William G.

doi:10.1074/jbc.m408026200

Cited by 122 publications

(118 citation statements)

References 52 publications

(67 reference statements)

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“…Recently, the checkpoint kinase ataxia telangiectasia and Rad3-related was shown to interact with Mcm7 through its regulatory subunit ATRIP and to phosphorylate Mcm2 specifically at Ser-108, suggesting that MCM is a target of the S-phase checkpoint pathway (14,15). This is consistent with the finding that Ser-108 Mcm2 phosphorylation is increased in cells challenged with DNA-damaging agents.…”

supporting

confidence: 81%

“…This is consistent with the finding that Ser-108 Mcm2 phosphorylation is increased in cells challenged with DNA-damaging agents. However, because basal levels of Mcm2 Ser-108 phosphorylation are detected in the absence of damage or in cells in which ATR activity is impaired, it is likely that a different kinase exists that is able to phosphorylate Mcm2 at the same site (14,15). Finally, Mcm2 contains a canonical consensus site for casein kinase 2 (CK2), a kinase with pleiotropic functions that has also been implicated in regulating DNA replication proteins (16,17).…”

mentioning

confidence: 99%

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Identification of Mcm2 Phosphorylation Sites by S-phase-regulating Kinases

Montagnoli

Valsasina

Brotherton³

et al. 2006

Journal of Biological Chemistry

199

214

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Minichromosome maintenance 2-7 proteins play a pivotal role in replication of the genome in eukaryotic organisms. Upon entry into S-phase several subunits of the MCM hexameric complex are phosphorylated. It is thought that phosphorylation activates the intrinsic MCM DNA helicase activity, thus allowing formation of active replication forks. Cdc7, Cdk2, and ataxia telangiectasia and Rad3-related kinases regulate S-phase entry and S-phase progression and are known to phosphorylate the Mcm2 subunit. In this work, by in vitro kinase reactions and mass spectrometry analysis of the products, we have mapped phosphorylation sites in the N terminus of Mcm2 by Cdc7, Cdk2, Cdk1, and CK2. We found that Cdc7 phosphorylates Mcm2 in at least three different sites, one of which corresponds to a site also reported to be phosphorylated by ataxia telangiectasia and Rad3-related. Three serine/proline sites were identified for Cdk2 and Cdk1, and a unique site was phosphorylated by CK2. We raised specific anti-phosphopeptide antibodies and found that all the sites identified in vitro are also phosphorylated in cells. Importantly, although all the Cdc7-dependent Mcm2 phosphosites fluctuate during the cell cycle with kinetics similar to Cdc7 kinase activity and Cdc7 protein levels, phosphorylation of Mcm2 in the putative cyclin-dependent kinase (Cdk) consensus sites is constant during the cell cycle. Furthermore, our analysis indicates that the majority of the Mcm2 isoforms phosphorylated by Cdc7 are not stably associated with chromatin. This study forms the basis for understanding how MCM functions are regulated by multiple kinases within the cell cycle and in response to external perturbations.

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supporting

confidence: 81%

mentioning

confidence: 99%

Identification of Mcm2 Phosphorylation Sites by S-phase-regulating Kinases

Montagnoli

Valsasina

Brotherton³

et al. 2006

Journal of Biological Chemistry

199

214

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“…MCMs proteins have been shown to be direct targets of ATM/ATR kinases (Cortez et al, 2004;Ishimi et al, 2003;Shi et al, 2007;Yoo et al, 2004), suggesting that the MCMs may be targets or effectors of the replication checkpoint. It has been shown that the phosphorylation of MCM4 in the checkpoint control inhibits DNA replication, which includes blockage of DNA fork progression through inactivation of the MCM complex (Ishimi et al, 2003).…”

Section: Is Udu Required For Dna Replication?mentioning

confidence: 99%

“…Hence, Udu in udu tu24 mutants, which lacks both PAH-L and SANT-L domains, may be unable to interact with MCM proteins, probably leading to increased DNA damage, indicated by our comet assay. The reduction of BrdU-positive cells in udu tu24 mutants further supports our notion that progression though the S phase is delayed or prevented.MCMs proteins have been shown to be direct targets of ATM/ATR kinases (Cortez et al, 2004;Ishimi et al, 2003;Shi et al, 2007;Yoo et al, 2004), suggesting that the MCMs may be targets or effectors of the replication checkpoint. It has been shown that the phosphorylation of MCM4 in the checkpoint control inhibits DNA replication, which includes blockage of DNA fork progression through inactivation of the MCM complex (Ishimi et al, 2003).…”

mentioning

confidence: 99%

Udu Deficiency Activates DNA Damage Checkpoint

Lim

Chong

Jiang

2009

MBoC

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Udu has been shown to play an essential role during blood cell development; however, its roles in other cellular processes remain largely unexplored. In addition, ugly duckling (udu) mutants exhibited somite and myotome boundary defects. Our fluorescence-activated cell sorting analysis also showed that the loss of udu function resulted in defective cell cycle progression and comet assay indicated the presence of increased DNA damage in udu tu24 mutants. We further showed that the extensive p53-dependent apoptosis in udu tu24 mutants is a consequence of activation in the Atm-Chk2 pathway. Udu seems not to be required for DNA repair, because both wild-type and udu embryos similarly respond to and recover from UV treatment. Yeast two-hybrid and coimmunoprecipitation data demonstrated that PAH-L repeats and SANT-L domain of Udu interacts with MCM3 and MCM4. Furthermore, Udu is colocalized with 5-bromo-2 -deoxyuridine and heterochromatin during DNA replication, suggesting a role in maintaining genome integrity. INTRODUCTIONugly duckling (udu tu24 ) mutant was first isolated from the 1996 Tü bingen screen and has been shown to exhibit a short body-axis with massive cell death (Hammerschmidt et al., 1996). Another udu allele, udu sq1 , was isolated in a genetic screen aiming at mutants with defects in hematopoiesis (Liu et al., 2007). Positional cloning revealed that Udu protein encodes a novel nuclear factor consisting of three conserved regions (CR-1, CR-2, and CR-3) that do not share similarity with any known domains, two paired amphipathic ␣-helix like (PAH-L) repeats, and one putative SW13, ADA2, N-Cor and TFIIIB like (SANT-L) domain. The C-terminal PAH-L and SANT-L domains have been shown to be essential in primitive erythroid cell development (Liu et al., 2007). The PAH domain, first identified in yeast SIN3 (Wang et al., 1990), has been demonstrated to mediate protein-protein interactions (Spronk et al., 2000). SIN3 has no intrinsic DNA binding ability and has to be targeted to gene promoters by interacting with DNA binding proteins, thereafter positively and negatively regulating genes involved in diverse cellular functions (Silverstein and Ekwall, 2005). The SANT domain is a highly conserved motif with similarity to Myb DNA binding domain (Aasland et al., 1996), which has been shown to be critical in regulating chromatin accessibility (Boyer et al., 2002(Boyer et al., , 2004.The DNA damage response pathway is a cellular surveillance system that senses the presence of damaged DNA and elicits checkpoint activation and subsequent lesion repair in preventing amplification or loss of genes or chromosomes (Zhou and Elledge, 2000). The critical components of DNA damage checkpoint are two phosphatidyl inositol 3-kinaselike kinase family proteins: Ataxia telangiectasia mutated (ATM) and ATM-and Rad3-related (ATR) (Abraham, 2003;Shiloh, 2003). ATR functions as a sensor and transducer in response to UV light and presumably to genotoxic agents that give rise to stalled replication forks, and subsequently activates Checkp...

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“…We next tested whether Dpb11 could function as a Mec1 activator. We immunopurified Mec1-Ddc2 from yeast lysates and incubated the complexes with recombinant Dpb11 and an established substrate, MCM2 (22,23). Addition of Dpb11 strongly stimulated the kinase activity of Mec1 in a dose-dependent manner (Fig.…”

Section: Dpb11mentioning

confidence: 99%

Dpb11 activates the Mec1–Ddc2 complex

Mordes

Nam

Chen

2008

Proc. Natl. Acad. Sci. U.S.A.

112

116

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The Saccharomyces cerevisiae Mec1-Ddc2 checkpoint kinase complex (the ortholog to human ATR-ATRIP) is an essential regulator of genomic integrity. The S. cerevisiae BRCT repeat protein Dpb11 functions in the initiation of both DNA replication and cell cycle checkpoints. Here, we report a genetic and physical interaction between Dpb11 and Mec1-Ddc2. A C-terminal domain of Dpb11 is sufficient to associate with Mec1-Ddc2 and strongly stimulates the kinase activity of Mec1 in a Ddc2-dependent manner. Furthermore, Mec1 phosphorylates Dpb11 and thereby amplifies the stimulating effect of Dpb11 on Mec1-Ddc2 kinase activity. Thus, Dpb11 is a functional ortholog of human TopBP1, and the Mec1/ATR activation mechanism is conserved from yeast to humans.

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Mcm2 Is a Direct Substrate of ATM and ATR during DNA Damage and DNA Replication Checkpoint Responses

Cited by 122 publications

References 52 publications

Identification of Mcm2 Phosphorylation Sites by S-phase-regulating Kinases

Identification of Mcm2 Phosphorylation Sites by S-phase-regulating Kinases

Udu Deficiency Activates DNA Damage Checkpoint

Dpb11 activates the Mec1–Ddc2 complex

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