Role of regulation of eukaryotic DNA replication in preserving genomic stability

描述（由申请人提供）：真核细胞生物学的核心原则是DNA复制必须严格控制，以便每个细胞周期只发生一次。据推测，这种控制对保持基因组完整性至关重要，但在很大程度上未经检验。我们的长期目标是了解如何在真核生物基因组中分散的数千个复制起点可靠地阻止DNA复制的重新启动，并辨别破坏这种控制对基因组稳定性的影响。我们在出芽酵母中研究复制控制，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，这些机制需要以如此非凡的保真度实现这种控制。此外，在出芽酵母中可用的分子遗传工具使我们能够应用简单和复杂的技术来查询破坏复制控制的影响。在之前的资助期内，我们证明了周期蛋白依赖性激酶（CDKs）使用多种重叠机制来防止单个细胞周期内起源的重新启动。我们还表明，由于失去这些控制而引起的再复制导致显著的染色体断裂和致死率，为复制控制的重要性提供了以前未知的理由。最近，我们提供了第一个证据，证明再复制是诱导基因扩增的高效手段（Green等，Science, in press）。再复制诱导的基因扩增（RRIGA）发生的效率非常高（大约1/20的再起始事件）。这一发现支持了一个令人信服的假设，即即使是复制控制的轻微损伤也可能导致基因组不稳定。最终，我们希望证明再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的机制以及进一步研究复制控制缺失的生物学后果和意义，来扩大我们对复制控制缺失如何导致基因组不稳定的理解。我们建议：(1)定义实现RRIGA的机制和参数；(2)确定局部调控因子如何调节高度易发起始点的复制控制；(3)确定RRIGA是否参与进化适应模式；(4)确定再复制是否会诱导染色体错分离。这些数据将大大增强我们对重复复制如何促进基因组不稳定性的理解，并深入了解复制控制缺失的生物学意义。

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.