Abstract
Two checkpoint pathways have been described in fission yeast: the intra-S phase checkpoint that is triggered by stalled replication forks and requires the Cds1 protein as the main effector kinase, and the G2/M checkpoint that is activated by DNA damage and requires the Chk1 kinase. Both pathways prevent untimely entry into mitosis, delaying cell cycle progression until DNA damage is repaired. Previously, our laboratory showed that a mutant defective in polymerase ε (Cdc20) requires Chk1, but not Cds1, to maintain cell viability. I set out to test whether other replication mutants are sensitive to the loss of Chk1 or Cds1, and my results suggested that mutants defective in DNA replication initiation are sensitive to loss of Chk1, whilst mutations that block DNA replication elongation are sensitive to loss of Cds1. To confirm that the Chk1-sensitivity of these mutants was specific to defects in DNA replication initiation, we conducted a genetic screen for cell cycle mutants that require Chk1, but not Cds1 to maintain viability when grown at semi-permissive temperatures. This screen led to the identification of two mutants, rid1-1 and rid2-1, that encode two proteins essential for initiation of DNA replication, Orp1 and Mcm4, thus confirming our hypothesis that mutants defective in DNA replication initiation display a chk1-dependent, cds1-independent phenotype. Eukaryotic DNA replication requires that DNA synthesis occurs on chromatin-bound DNA templates. Several proteins have been identified that interact directly with chromatin and are important for normal chromosomal structure and function. In S. pombe, three proteins, Abp1, and the closely related Cbh1 and Cbh2 are homologous to the human centromere-binding protein CENP-B, and have been implicated in the assembly of centromeric heterochromatin. Fission yeast cells lacking Abp1 show an increase in mini-chromosome instability suggesting that Abp1 is important for chromosome segregation and/or DNA synthesis. Using a two-hybrid assay, our lab showed that Abp1 interacts with the DNA replication protein Cdc23 (MCM10). In support to the yeast two hybrid data, I have found that the ∆abp1 mutant displays a synthetic phenotype with a cdc23 temperature-sensitive mutant. Moreover, genetic interactions were also observed between abp1+ and four additional DNA replication initiation genes cdc18+, cdc21+, orc1+, and orc2+. Interestingly, we find that S phase is delayed in cells deleted for abp1+ when released from a G1 block. However, no delay is observed when cells are released from an early S phase arrest induced by hydroxyurea suggesting that Abp1 functions prior to, or coincident with, the initiation of DNA replication.