A central tenet of eukaryotic cell biology is that DNA replication must be tightly controlled so that it occurs only once per cell cycle. We study replication control in budding yeast because this model system offers an exceptional opportunity to dissect the complex, overlapping mechanisms that are required to achieve this control with such extraordinary fidelity. Additionally, the molecular genetic tools available in budding yeast allow us to apply both simple and sophisticated technologies to query the effects of disrupting replication control. Our longstanding work has been instrumental in revealing multiple overlapping mechanisms used by eukaryotes to reduce the probability of re-initiation within a cell cycle. However, since eukaryotic genomes contains thousands of replication origins, it may be impossible to eliminate rare, sporadic re-initiation over the course of multiple cell divisions, and this problem can be significantly exacerbated by just a slight compromise in replication control. Thus one of our long-term goals is to discern the role of re-replication as a driver of genome plasticity and instability. Recently we have shown that loss of replication control is a powerful instigator of genetic alteration and variation in eukaryotes. We find that inappropriate re-initiation of DNA replication within a cell cycle can induce tandem intrachromosomal amplifications and aneuploidy by up to 104-106 folds. Here we propose to (1) investigate the mechanism of re-replication induced gene amplification; (2) investigate the mechanism of re- replication induced aneuploidy; (3) expand our ability to detect re-replication and genetic alterations arising from re-replication; and (4) explore whether re-replication provides a source of spontaneous segmental amplification during normal cellular growth. This work will establish re-replication as a potential new source of genetic variation and alteration in budding yeast that could ultimately impact how we think about molecular evolution and oncogenesis.
Our studies in budding yeast suggest that the unscheduled re-replication of DNA could be a source of genetic alterations. Our proposed project will investigate how re-replication can cause two specific types of alterations frequently seen in cancer, gene amplification and chromosome aneuploidy, and explores whether re-replication could also contribute to the gene duplication critical for molecular evolution.
