The intra-S-phase checkpoint in fission yeast
MetadataShow full item record
Eukaryotic cells slow their progression through S phase upon DNA damage. The mechanism that leads to this slowing is called the intra-S-phase checkpoint. We wanted to (a) identify the molecular components of this checkpoint in the fission yeast, Schizosaccharomyces pombe , and (b) investigate the mechanism by which the slowing of S phase is achieved in this yeast. We used the methylating agent, methyl methane sulfonate (MMS), to introduce DNA damage. Our studies demonstrated that, in fission yeast, the intra-S-phase checkpoint is mediated by a pathway that includes Rad3 (similar to ATR and ATM in humans and Mec1 in budding yeast) and Cds1 (similar to Chk1 and Chk2 in humans and Rad53 in budding yeast). We also obtained evidence that a major downstream target of this pathway is the cyclin-dependent kinase, Cdc2. Using neutral-neutral 2D-gel analysis of replication intermediates, we found that retardation of early-origin firing and reduction in the rate of replication-fork movement seem to be responsible for the slowing of S phase upon DNA damage in wild type cells. Deletion of cds1 or dysregulation of the cyclin-dependent kinase, Cdc2, abolished the DNA-damage-induced origin inhibition and the replication-fork slowdown. Our observations in fission yeast provide complementary information to studies in budding yeast and vertebrates. In budding yeast, MMS-induced DNA damage leads to inhibition of late-firing origins, in a pathway dependent upon Mec1 and Rad53. In budding yeast, events downstream of Rad53 are less well understood, and the cyclin-dependent kinase, Cdc28, does not seem to be involved in regulation of late origins. In vertebrates, the intra-S-phase checkpoint requires phosphorylation of the S-phase specific cyclin-dependent kinase Cdk2 in an ATR- and/or ATM- and Chk1- and/or Chk2-dependent pathway to bring about retardation of S phase. Thus our studies indicate that the fission yeast intra-S-phase checkpoint mechanism is similar to that of vertebrates. Our genetic analyses suggested that, in fission yeast, the intra-S-phase checkpoint pathway may be identical to the S dNTP -M checkpoint pathway. The S dNTP -M checkpoint prevents mitosis when replication forks are stalled (frequently by treatment of cells with hydroxyurea (HU), which leads to depletion of dNTP pools by inhibition of ribonucleotide reductase). The apparent identity between these two pathways suggested that the initial event leading to a checkpoint signal should be similar or identical in the two cases. Stalled replication forks, which would be generated either by dNTP pool depletion or by methylated DNA, could be the common initial event for these checkpoints. If treatment with HU triggers the same checkpoint pathway as treatment with MMS, then how can one explain the fact that early origins fire and late origins are suppressed in HU-treated fission yeast cells, while early origins are retarded and late origins fire in MMS-treated cells? We suggest that late origin firing may require the availability of replication proteins released from early replication forks that have completed replication. Since all replication forks stop in HU-treated cells (and thus do not complete replication), but most forks only slow down (without stopping) in MMS-treated cells, late origin firing is prevented in HU-treated cells but permitted in MMS-treated cells.