title: Mcm2 phosphorylation and the response to replicative stress.
abstract: BACKGROUND
  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. 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.
  
  
  RESULTS
  We 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.
  
  
  CONCLUSIONS
  Together 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.
authors.affiliation: Department of Biochemistry, Schulich School of Medicine & Dentistry, University of Western Ontario, London, ON, N6A 5C1, Canada.
authors.forename: Brent E
authors.initials: BE
authors.lastname: Stead
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Brandl
authors.forename: Matthew K
authors.initials: MK
authors.lastname: Sandre
authors.forename: Megan J
authors.initials: MJ
authors.lastname: Davey
mesh_terms: D000483:Alleles; D026923:Amino Acid Transport Systems, Basic / Q000235:genetics / Q000378:metabolism; D002110:Caffeine / Q000494:pharmacology; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism; D064447:Checkpoint Kinase 2; D002868:Chromosomal Proteins, Non-Histone / Q000235:genetics / Q000378:metabolism*; D004249:DNA Damage; D004261:DNA Replication / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005472:Fluorouracil / Q000494:pharmacology; D017353:Gene Deletion; D006918:Hydroxyurea / Q000494:pharmacology; D009154:Mutation; D018384:Oxidative Stress; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D026923:Amino Acid Transport Systems, Basic; C502212:CAN1 protein, S cerevisiae; D018797:Cell Cycle Proteins; D002868:Chromosomal Proteins, Non-Histone; D004791:Enzyme Inhibitors; D029701:Saccharomyces cerevisiae Proteins; D002110:Caffeine; C489404:CDC7 protein, S cerevisiae; D064447:Checkpoint Kinase 2; D017346:Protein Serine-Threonine Kinases; C067022:RAD53 protein, S cerevisiae; C069199:MCM2 protein, S cerevisiae; D005472:Fluorouracil; D006918:Hydroxyurea