Role of regulation of eukaryotic DNA replication in preserving genomic 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。据推测，但在很大程度上未经检验，这种控制对保持基因组的完整性至关重要。我们的长期目标是了解如何在散布在真核生物基因组中的数千个复制起点可靠地防止DNA复制的重新启动，并了解破坏这种控制对基因组稳定性的影响。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。在之前的资助阶段，我们证明了细胞周期蛋白依赖性蛋白激酶(CDK)使用多种重叠机制来防止起源在单个细胞周期内重新启动。我们还表明，由于失去这些控制而产生的重新复制会导致显著的染色体断裂和致命性，为复制控制的重要性提供了一个以前未知的理由。最近，我们提供了第一个证据，证明重新复制是一种高效的诱导基因放大的方法(Green等人，科学，在出版中)。重新复制诱导的基因扩增(RRIGA)以非凡的效率发生(大约1/20的重新启动事件)。这一发现支持了一个令人信服的假设，即即使复制控制的微小损害也可能导致基因组的不稳定。最终，我们希望证明重新复制可以驱动在肿瘤发生、人类遗传变异和进化中观察到的拷贝数变化。在这里，我们建议通过探索RRIGA背后的机制以及进一步研究复制控制丧失的生物学后果和意义来扩大我们对复制控制丧失如何导致基因组不稳定的理解。我们建议(1)定义使RRIGA有效的机制和参数；(2)确定本地调节因子如何在高度容易重新启动的起始处调节复制控制；(3)确定RRIGA是否参与进化适应模型；以及(4)确定重新复制是否会导致染色体错误分离。这些数据将大大增强我们对重新复制如何促进基因组不稳定的理解，以及对复制控制丧失的生物学意义的洞察。

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. It is presumed, but largely untested, that this control is vital for preserving genome integrity. Our long-term goal is to understand how the re-initiation of DNA replication is reliably prevented at the thousands of replication origins scattered throughout eukaryotic genomes, and to discern the effect of disrupting this control on genome stability. 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 controls. In previous funding periods, we demonstrated that cyclin-dependent kinases (CDKs) use multiple overlapping mechanisms to prevent origins from re-initiating within a single cell cycle. We have also shown that re-replication arising from loss of these controls leads to significant chromosomal breakage and lethality, providing a previously unknown justification for the importance of replication control. More recently, we provided the first evidence that re-replication is a highly efficient means to induce gene amplification (Green et al, Science, in press). Re-replication induced gene amplification (RRIGA) occurred with extraordinary efficiency (roughly 1/20 re-initiation events). This finding supports the compelling hypothesis that even minor impairment of replication control may contribute to genome instability. Ultimately, we hope to demonstrate that re-replication can drive the copy number changes observed in tumorigenesis, human genetic variation, and evolution. Here we propose to expand our understanding of how the loss of replication control leads to genomic instability, both by probing the mechanisms that underlie RRIGA as well as by further investigating the biological consequences and significance of loss of replication control. We propose to (1) define the mechanism and parameters enabling RRIGA; (2) determine how local regulatory factors modulate replication control at origins that are highly susceptible to re-initiation; (3) determine whether RRIGA participates in a model of evolutionary adaptation; and (4) establish whether re-replication can induce chromosome missegregation. These data will significantly enhance our understanding of how re-replication promotes genomic instability, as well as give insight into the biological significance of loss of replication control.