Identification of MCM4 as a Target of the DNA Replication Block Checkpoint System

Abstract

Inhibition of the progression of DNA replication prevents further initiation of DNA replication and allows cells to maintain arrested replication forks, but the proteins that are targets of the replication checkpoint system remain to be identified. We report here that human MCM4, a subunit of the putative DNA replicative helicase, is extensively phosphorylated in HeLa cells when they are incubated in the presence of inhibitors of DNA synthesis or are exposed to UV irradiation. The data presented here indicate that the consecutive actions of ATR-CHK1 and CDK2 kinases are involved in this phosphorylation in the presence of hydroxyurea. The phosphorylation sites in MCM4 were identified using specific anti-phosphoantibodies. Based on results that showed that the DNA helicase activity of the MCM4-6-7 complex is negatively regulated by CDK2 phosphorylation, we suggest 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.The initiation of DNA replication in eukaryotes is triggered by activation of the MCM 1 (2-7) complex, which is loaded with CDC6 and CDT1 onto replication origins to which ORCs are bound (1-3). The activation of the MCM complex requires phosphorylation of the MCM2 subunit by CDC7/DBF4 kinase and association of CDC45 with the origin region after the action of cyclin-dependent kinase. The MCM2-7 complex is the most likely candidate to act as the DNA replication helicase that catalyzes the unwinding of the DNA duplex during replication (4, 5). Although all MCM subunits possess a DNA-dependent ATPase motif in their central domain, DNA helicase activity is detected only with the MCM4-6-7 complex, which dimerizes to form a hexamer (6 -9). Thus, it is possible that the MCM4-6-7 hexamer is an activated form of the MCM (2-7) hexamer, although other mechanisms leading to the activation of the MCM helicase activity have been proposed (10). MCM4 is phosphorylated in vivo at least in part by cyclin-dependent kinases, which probably leads to the inactivation of the MCM complex (11, 12). We reported previously (13) that the DNA helicase activity of the MCM4-6-7 complex is inhibited by the sitespecific phosphorylation of MCM4 with CDK2-cyclin A. In Saccharomyces cerevisiae, it has been shown that cyclin-dependent kinases play a role leading to the exclusion of MCM4 from the nucleus (14). Other targets of the cyclin-dependent kinase activity that contribute to the negative regulation of DNA replication include ORC, CDC6, and CDT1 (15).Cells normally protect the integrity of their genome from stresses such as ultraviolet light, ionizing radiation, alkylating reagents, and DNA replication blockage (16 -21). Treatment with hydroxyurea (HU), which inhibits ribonucleotide reductase, not only blocks the progression of DNA replication but also activates a DNA replication checkpoint system that is required to maintain genomic integrity. In the presence of HU or methyl methanesulfonate, the initiation of DNA ...

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

“…9). In human cells, MCM4 also undergoes caffeine-sensitive, checkpointdependent phosphorylation, but a direct role for ATR was not addressed (37). While this manuscript was in preparation, it was reported that ATM and ATR phosphorylate mammalian MCM2 on a site analogous to the one that we have found in Xmcm2 (38 (lanes 1 and 2), geminin ( lanes 3 and 4), or caffeine (lanes 5 and 6).…”

Udu Deficiency Activates DNA Damage Checkpoint

“…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).…”

“…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). Thus, the DNA sequence fidelity, together with associated chromatin structure must be accurately replicated to maintain genetic and epigenetic information through cell generations.…”

“…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).…”

Minichromosome maintenance proteins are direct targets of the ATM and ATR checkpoint kinases

“…The retinoblastoma protein, cyclin D, and papillomavirus E6 protein also bind to MCM7 (22)(23)(24). The MCM complex is also an indirect target of checkpoint signaling via modulation of cyclin-dependent kinase-mediated MCM4 phosphorylation (25).…”