Abstract
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.
Highlights
The 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
Growth of mcm2AA and mcm2EE cells in the presence of replicative stress We examined the growth of the mcm2AA strain on media containing agents that cause replicative stress (Figure 1A & B)
The mcm2AA strain grows poorly upon constant exposure to the ribonucleotide reductase inhibitor, hydroxyurea (HU), which interferes with the integrity of DNA replication forks and induces an S phase checkpoint [28,29]
Summary
The 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. The catalytic core of the replicative helicase in eukaryotic cells is a heterohexameric complex comprised of the minichromosome maintenance proteins 2 through 7 (Mcm2-7; [9,10]). Mcm functions within the CMG complex comprised of Cdc, Mcm and the tetrameric GINS complex (Sld, Psf, Psf and Psf3) [11,12]. The catalytic activity for DNA unwinding resides in Mcm with Cdc and GINS playing roles in limiting Mcm activity to S phase and providing scaffolding functions within the replisome [11,15,16,17]. CMG is isolated from replicating yeast cells as part of the RPC (replisome progression complex) that includes the checkpoint protein Mrc, the fork pausing complex Tof1-Csm, the histone chaperone FACT and the sister chromatid cohesion factor, Ctf4 [16]
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