Eukaryotic Completion of DNA Replication

项目摘要 细胞识别和完成复制区域的机制， 倍增点必须非常有效，每次细胞分裂沿着发生数千次 人类的染色体，是所有细胞中基因组稳定性所需的基本步骤。 然而，直到最近，这一过程如何发生的问题还没有被描述，在任何细胞中 类型.我们最近证明了在E.大肠杆菌涉及 当达到其“倍增点”时有效限制细胞复制的酶系统 通过允许会聚复制叉在过度、过度 复制的区域被切开、切除和连接。完成反应需要RecBCD 并涉及与修复双链断裂相关的几种蛋白质，包括SbcC- SbcD-ExoI.然而，与双链断裂修复不同的是，完成独立于 同源重组和RecA。 完成DNA复制所需的许多细菌蛋白质在DNA复制中有明确的同源物。 真核生物细菌SbcC-SbcD和ExoI与Mre 11-Rad 50和CtIP高度保守， 对基因组稳定性、正常发育和 哺乳动物的生存能力。在这里，我们建议将这些重要的发现扩展到真核细胞， 并确定催化模型中复制完成的酶途径 真核生物，酿酒酵母。我们采用了一种新的方法，使我们能够识别， 绘制并表征复制直接在真核染色体上完成的位点。 我们将使用这种方法来确定Mre 11-Rad 50-CtIP在细胞凋亡过程中的重要作用。 复制，确定真核生物完成反应所需的酶，并鉴定 完成突变体中的合成致死基因，可以作为潜在治疗的靶点。的 这些研究的结果将确定一个基本的，但迄今尚未研究的，细胞的方面， 复制是基因组稳定性的核心。

Project Summary The mechanism by which cells recognize and complete replicated regions at their precise doubling point must be remarkably efficient, occurring thousands of times per cell division along the chromosomes of humans, and is a fundamental step required for genomic stability in all cells. Yet until recently, the question of how this process occurs had not been characterized, in any cell type. We recently demonstrated that the completion of DNA replication in E. coli involves an enzymatic system that effectively limits cellular replication when it reaches its `doubling point' by allowing converging replication forks to transiently pass each other before the excess, over- replicated regions are incised, resected, and joined. The completion reaction requires RecBCD and involves several proteins associated with repairing double-strand breaks including, SbcC- SbcD-ExoI. However, unlike double-strand break repair, completion occurs independently of homologous recombination and RecA. Many of bacterial proteins required to complete DNA replication have clear homologs in eukaryotes. Bacterial SbcC-SbcD and ExoI are highly conserved with Mre11-Rad50 and CtIP, a poorly characterized nuclease complex essential for genome stability, normal development, and viability in mammals. Here, we propose to extend these important findings to eukaryotic cells, and to determine the enzymatic pathway that catalyzes the completion of replication in the model eukaryote, Saccharomyces cerevisiae. We employ a novel approach that enables us to identify, map, and characterize sites where replication completes directly on eukaryotic chromosomes. We will use this approach to establish the essential role of Mre11-Rad50-CtIP during cellular replication, determine the enzymes required for the eukaryotic completion reaction, and identify synthetic lethal genes in completion mutants, that can be targeted for potential therapeutics. The results of these studies will identify a fundamental, yet heretofore unstudied, aspect of cellular replication that is central to genome stability.