Role of regulation of eukaryotic DNA replication in preserving genomic stability

描述（由申请人提供）：真核细胞生物学的中心原则是DNA复制必须受到严格控制，以便每个细胞周期仅发生一次。据推测，但在很大程度上未经测试，这种控制是至关重要的，以保持基因组的完整性。我们的长期目标是了解如何在分散在真核基因组中的数千个复制起点处可靠地防止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。我们研究芽殖酵母的复制控制，因为这个模型系统提供了一个特殊的机会，解剖复杂的，重叠的机制，需要实现这种控制与这种非凡的保真度。此外，芽殖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。在以前的资助期间，我们证明了细胞周期蛋白依赖性激酶（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，这些控制的损失所产生的再复制导致显着的染色体断裂和致死性，提供了一个以前未知的理由复制控制的重要性。最近，我们提供了第一个证据，即再复制是诱导基因扩增的高效手段（绿色等人，科学，出版中）。再复制诱导的基因扩增（RRIGA）以非凡的效率发生（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，即使是轻微的损害复制控制可能有助于基因组的不稳定性。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，我们建议扩大我们的理解如何复制控制的损失导致基因组的不稳定性，无论是通过探测RRIGA的基础机制，以及通过进一步调查的生物学后果和复制控制的损失的意义。我们建议：（1）定义机制和参数，使RRIGA;（2）确定本地调节因子如何调节复制控制的起点，是高度敏感的重新启动;（3）确定是否RRIGA参与了一个模型的进化适应;（4）建立是否重新复制可以诱导染色体错误分离。这些数据将显著增强我们对再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丧失的生物学意义。

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.