Checkpoint-dependent regulation of origin firing and replication fork movement in response to DNA damage in fission yeast.
To elucidate the checkpoint mechanism responsible for slowing passage through S phase when fission yeast cells are treated with the DNA-damaging agent methyl methanesulfonate (MMS), we carried out two-dimensional gel analyses of replication intermediates in cells synchronized by cdc10 block (in G(1)) followed by release into synchronous S phase. The results indicated that under these conditions early-firing centromeric origins were partially delayed but late-firing telomeric origins were not delayed. Replication intermediates persisted in MMS-treated cells, suggesting that replication fork movement was inhibited. These effects were dependent on the Cds1 checkpoint kinase and were abolished in cells overexpressing the Cdc25 phosphatase, suggesting a role for the Cdc2 cyclin-dependent kinase. We conclude that both partial inhibition of the firing of a subset of origins and inhibition of replication fork movement contribute to the slowing of S phase in MMS-treated fission yeast cells.
Kumar
Sanjay
S
Department of Cancer Biology, Roswell Park Cancer Institute, Elm & Carlton Streets, Buffalo, New York 14263-0001, USA.
Huberman
Joel A
JA
D018797:Cell Cycle Proteins; D064447:Checkpoint Kinase 2; D004249:DNA Damage; D015180:Electrophoresis, Gel, Two-Dimensional; D008741:Methyl Methanesulfonate / Q000494:pharmacology; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D018741:Replication Origin / Q000187:drug effects; D016196:S Phase / Q000187:drug effects; D012568:Schizosaccharomyces / Q000187:drug effects / Q000235:genetics* / Q000254:growth & development*; D029702:Schizosaccharomyces pombe Proteins / Q000378:metabolism; D016615:Telomere / Q000187:drug effects / Q000378:metabolism; D014157:Transcription Factors; D020687:cdc25 Phosphatases / Q000378:metabolism
D018797:Cell Cycle Proteins; D029702:Schizosaccharomyces pombe Proteins; D014157:Transcription Factors; C061178:cdc10 protein, S pombe; D008741:Methyl Methanesulfonate; D064447:Checkpoint Kinase 2; C578636:Cds1 protein, S pombe; D017346:Protein Serine-Threonine Kinases; D020687:cdc25 Phosphatases