The direct binding of Mrc1, a checkpoint mediator, to Mcm6, a replication helicase, is essential for the replication checkpoint against methyl methanesulfonate-induced stress.
Mrc1 plays a role in mediating the DNA replication checkpoint. We surveyed replication elongation proteins that interact directly with Mrc1 and identified a replicative helicase, Mcm6, as a specific Mrc1-binding protein. The central portion of Mrc1, containing a conserved coiled-coil region, was found to be essential for interaction with the 168-amino-acid C-terminal region of Mcm6, and introduction of two amino acid substitutions in this C-terminal region abolished the interaction with Mrc1 in vivo. An mcm6 mutant bearing these substitutions showed a severe defect in DNA replication checkpoint activation in response to stress caused by methyl methanesulfonate. Interestingly, the mutant did not show any defect in DNA replication checkpoint activation in response to hydroxyurea treatment. The phenotype of the mcm6 mutant was suppressed when the mutant protein was physically fused with Mrc1. These results strongly suggest for the first time that an Mcm helicase acts as a checkpoint sensor for methyl methanesulfonate-induced DNA damage through direct binding to the replication checkpoint mediator Mrc1.
Komata
Makiko
M
Laboratory of Chromosome Structure and Function, Department of Biological Science, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Yokohama City, Kanagawa, Japan.
Bando
Masashige
M
Araki
Hiroyuki
H
Shirahige
Katsuhiko
K
D019943:Amino Acid Substitution / Q000187:drug effects; D018797:Cell Cycle Proteins / Q000737:chemistry / Q000378:metabolism*; D002868:Chromosomal Proteins, Non-Histone / Q000737:chemistry / Q000378:metabolism*; D004261:DNA Replication / Q000187:drug effects*; D006918:Hydroxyurea / Q000494:pharmacology; D008741:Methyl Methanesulfonate / Q000633:toxicity*; D064151:Minichromosome Maintenance Complex Component 6; D009154:Mutation / Q000235:genetics; D011485:Protein Binding / Q000187:drug effects; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000187:drug effects* / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects; D013312:Stress, Physiological / Q000187:drug effects*
D018797:Cell Cycle Proteins; D002868:Chromosomal Proteins, Non-Histone; C441579:MRC1 protein, S cerevisiae; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D008741:Methyl Methanesulfonate; C491207:MCM6 protein, S cerevisiae; D064151:Minichromosome Maintenance Complex Component 6; D006918:Hydroxyurea