Understanding the mechanisms that result in increased genetic instability is of great interest to those studying the development of cancer. Inhibition of DNA replication can lead to increases in the frequency of gross chromosomal rearrangements (GCRs), that can arise from DNA damage associated with stalled or collapsed replication forks. In the yeast, Schizosaccharomyces pombe, cells activate specific checkpoint pathways in response to DNA damage, and these checkpoints are essential to allow time for repair. We have discovered a novel checkpoint phenotype associated with mutants in DNA replication initiation proteins called rid (for replication initiation defective). When grown under semi-permissive conditions, rid mutants are delayed in the cell cycle but fail to activate the inter-S phase checkpoint. However, these cells still require the checkpoint kinase Chk1 for viability, suggesting an alternative checkpoint is activated. We propose to investigate the nature of the damage generated in rid mutants and to identify checkpoint/repair proteins required for rid viability. To do this, we will employ both genetic screens and gene microarray analysis. We will also determine whether mutation rates, including the incidence of GCRS, increase in the rid or checkpoint mutants. The results of the proposed experiments should provide a much better understanding of checkpoint pathways that respond to defects in the initiation of DNA replication. Considering the conserved aspects of many of these processes, our experimental findings are likely to be applicable to higher eukaryotic cells, including human. Moreover, our results will likely identify new drug targets for cancer therapy, and reveal potentially less mutagenic strategies to block cell cycle progression in cancer cells.
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