Dataset Information

Phosphorylation of MCM3 on Ser-112 regulates its incorporation into the MCM2-7 complex.

ABSTRACT: During late M and early G(1), MCM2-7 assembles and is loaded onto chromatin in the final step of prereplicative complex (pre-RC) formation. However, the regulation of MCM assembly remains poorly understood. Cyclin-dependent kinase (CDK)-dependent phosphorylation contributes to DNA replication by initially activating pre-RCs and subsequently inhibiting refiring of origins during S and M phases, thus limiting DNA replication to a single round. Although the precise roles of specific MCM phosphorylation events are poorly characterized, we now demonstrate that CDK1 phosphorylates MCM3 at Ser-112, Ser-611, and Thr-719. In vivo, CDK1-dependent phosphorylation of Ser-112 triggers the assembly of MCM3 with the remaining MCM subunits and subsequent chromatin loading of MCMs. Strikingly, loss of MCM3 triggers the destabilization of other MCM proteins, suggesting that phosphorylation-dependent assembly is essential for stable accumulation of MCM proteins. These data reveal that CDK-dependent MCM3 phosphorylation contributes to the regulated formation of the MCM2-7 complex.

SUBMITTER: Lin DI

PROVIDER: S-EPMC2430362 | biostudies-literature | 2008 Jun

REPOSITORIES: biostudies-literature

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Phosphorylation of MCM3 on Ser-112 regulates its incorporation into the MCM2-7 complex.

Lin Douglas I DI Aggarwal Priya P Diehl J Alan JA

Proceedings of the National Academy of Sciences of the United States of America 20080604 23

During late M and early G(1), MCM2-7 assembles and is loaded onto chromatin in the final step of prereplicative complex (pre-RC) formation. However, the regulation of MCM assembly remains poorly understood. Cyclin-dependent kinase (CDK)-dependent phosphorylation contributes to DNA replication by initially activating pre-RCs and subsequently inhibiting refiring of origins during S and M phases, thus limiting DNA replication to a single round. Although the precise roles of specific MCM phosphoryla ...[more]

PMID: 18524952

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Project description:BackgroundThe replicative helicase in eukaryotic cells is comprised of minichromosome maintenance (Mcm) proteins 2 through 7 (Mcm2-7) and is a key target for regulation of cell proliferation. In addition, it is regulated in response to replicative stress. One of the protein kinases that targets Mcm2-7 is the Dbf4-dependent kinase Cdc7 (DDK). In a previous study, we showed that alanine mutations of the DDK phosphorylation sites at S164 and S170 in Saccharomyces cerevisiae Mcm2 result in sensitivity to caffeine and methyl methanesulfonate (MMS) leading us to suggest that DDK phosphorylation of Mcm2 is required in response to replicative stress.ResultsWe show here that a strain with the mcm2 allele lacking DDK phosphorylation sites (mcm2AA) is also sensitive to the ribonucleotide reductase inhibitor, hydroxyurea (HU) and to the base analogue 5-fluorouracil (5-FU) but not the radiomimetic drug, phleomycin. We screened the budding yeast non-essential deletion collection for synthetic lethal interactions with mcm2AA and isolated deletions that include genes involved in the control of genome integrity and oxidative stress. In addition, the spontaneous mutation rate, as measured by mutations in CAN1, was increased in the mcm2AA strain compared to wild type, whereas with a phosphomimetic allele (mcm2EE) the mutation rate was decreased. These results led to the idea that the mcm2AA strain is unable to respond properly to DNA damage. We examined this by screening the deletion collection for suppressors of the caffeine sensitivity of mcm2AA. Deletions that decrease spontaneous DNA damage, increase homologous recombination or slow replication forks were isolated. Many of the suppressors of caffeine sensitivity suppressed other phenotypes of mcm2AA including sensitivity to genotoxic drugs, the increased frequency of cells with RPA foci and the increased mutation rate.ConclusionsTogether these observations point to a role for DDK-mediated phosphorylation of Mcm2 in the response to replicative stress, including some forms of DNA damage. We suggest that phosphorylation of Mcm2 modulates Mcm2-7 activity resulting in the stabilization of replication forks in response to replicative stress.

Dataset Information

Phosphorylation of MCM3 on Ser-112 regulates its incorporation into the MCM2-7 complex.

Publications

Phosphorylation of MCM3 on Ser-112 regulates its incorporation into the MCM2-7 complex.

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