Role of regulation of eukaryotic DNA replication in preserving genome stability

 描述(申请人提供)：真核细胞生物学的一个中心原则是必须严格控制DNA复制，使其在每个细胞周期中只发生一次。我们研究发芽酵母的复制控制，是因为这个模型系统提供了一个特殊的机会来剖析复杂的、重叠的机制，这些机制是实现这种控制所必需的，而且非常准确。此外，发芽酵母中可用的分子遗传工具允许我们应用简单和复杂的技术来质疑破坏复制控制的影响。我们长期的工作有助于揭示真核生物用来降低细胞周期内重新启动的可能性的多个重叠机制。然而，由于真核基因组包含数千个复制起点，因此可能不可能消除在多个细胞分裂过程中罕见的、零星的重新启动， 仅在复制控制方面稍有妥协，这个问题就会显著加剧。因此，我们的长期目标之一是辨别再复制作为基因组可塑性和不稳定性的驱动因素所起的作用。最近，我们已经证明，复制控制的丧失是真核生物基因改变和变异的强大诱因。我们发现，在一个细胞周期内不适当地重新启动DNA复制可以诱导串联的染色体内扩增和高达104-106倍的非整倍体。在这里，我们建议(1)研究再复制诱导基因扩增的机制；(2)研究再复制诱导非整倍体的机制；(3)扩大我们检测再复制和由再复制引起的遗传变化的能力；(4)探索再复制是否提供了正常细胞生长过程中自发节段性扩增的来源。这项工作将建立重新复制作为萌芽酵母遗传变异和改变的潜在新来源，这最终可能影响我们对分子进化和肿瘤发生的看法。

 DESCRIPTION (provided by applicant): 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.