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 ...

Mcm proteins that play an essential role in eukaryotic DNA replication are phosphorylated in vivo, and cyclindependent protein kinase is at least in part responsible for the phosphorylation of Mcm4. Our group reported that the DNA helicase activity of Mcm4,6,7 complex, which may be involved in initiation of DNA replication, is inhibited following phosphorylation by Cdk2/cyclin A in vitro. Here, we further examined the interplay between mouse Mcm4,6,7 complex and cyclin-dependent kinases and determined the sites required for the phosphorylation of Mcm4. Six Ser and Thr residues, in all, were required for the phosphorylation. Inhibition of Mcm4,6,7 helicase activity by Cdk2/cyclin A was largely relieved by introducing mutations in these residues of Mcm4. Anti-phosphothreonine antibodies raised against one of these sites reacted with Mcm4 prepared from HeLa cells at mitotic phase but did not bind to those at G 1 and G 1 /S, suggesting that this site is mainly phosphorylated in the mitotic phase. Mcm4,6,7 complex purified from HeLa cells at the mitotic phase exhibited a low level of DNA helicase activity, compared with the complexes prepared from cells at other phases. These results suggest that phosphorylation of Mcm4 at specific sites leads to loss of Mcm4,6,7 DNA helicase activity.

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