Regulation of DNA Replication and Repair

描述(申请人提供)：这项研究的长期目标是了解生物体如何通过协调其DNA复制机制与参与修复、损伤耐受和细胞周期进展的其他细胞蛋白质的行动来准确复制他们的遗传物质。如果生物体不能做到这一点，可能会产生从癌症等疾病到死亡的灾难性后果。拟议的研究计划利用生化和遗传方法来了解大肠杆菌复制DNA聚合酶的β加工能力钳在协调DNA复制、修复、损伤耐受性和细胞周期进展中所起的作用。β钳夹参与DNA损伤检查点控制和跨损伤DNA合成(TLS)。此外，它还与多种已知在DNA新陈代谢的各个方面起作用的蛋白质相互作用。作为一个目标，我们将检验我们的假设，即β与不同umuDC基因产物的独特相互作用影响复制、检查点和TLS之间的选择。这些研究将对理解复制和TLS在原核和真核细胞中的整合具有广泛的相关性。作为第二个目标，我们将通过确定dna N59β突变体对紫外线的敏感性的分子基础，来测试我们的建议，即β参与至少一个DNA修复和/或umuDC独立的损伤耐受功能。这些研究将进一步加深我们对复制和修复耦合机制的理解。作为第三个目标，我们将测试我们的假设，即某些伙伴蛋白质与Beta上的重叠表面结合，并且通过相互竞争与夹子结合，Beta能够调节哪些蛋白质获得复制叉子。这些研究将为聚合酶切换和复制分叉管理的基本机制提供重要的见解。综上所述，我们预计，对大肠杆菌中DNA复制、DNA损伤耐受和修复以及细胞周期进展的协调调控的研究将为理解人类的这些相同过程提供一个有价值的框架，因为在人类中，这些事件的复杂性要大得多。

DESCRIPTION (provided by applicant): The long-term goal of this research is to understand how organisms accurately duplicate their genetic material by coordinating the actions of their DNA replication machinery with those of other cellular proteins involved in repair, damage tolerance, and cell cycle progression. Failure of an organism to do so can have catastrophic consequences ranging from disease, such as cancer, to death. The proposed research program utilizes both biochemical and genetic approaches to understand the roles played by the beta processivity clamp of the E. coli replicative DNA polymerase in coordinating DNA replication, repair, damage tolerance, and cell cycle progression. The beta clamp participates in a DNA damage checkpoint control as well as in translesion DNA synthesis (TLS). In addition, it interacts with a variety of proteins known to function in various aspects of DNA metabolism. As one Aim, we will test our hypothesis that unique interactions of beta with the different umuDC gene products influence the choice between replication, checkpoint, and TLS. These studies will have broad relevance to understanding integration of replication and TLS in both prokaryotic and eukaryotic cells. As a second Aim, we will test our proposal that beta participates in at least one DNA repair and/or umuDC-independent damage tolerance function by determining the molecular basis of the UV light sensitivity of a dnaN59 beta mutant. These studies will further our understanding of mechanisms that couple replication and repair. As a third Aim, we will test our hypothesis that certain partner proteins bind to overlapping surfaces on beta, and that by competing with each other for binding to the clamp, beta is able to regulate which proteins gain access to the replication fork. These studies will provide important insights into fundamental mechanisms of polymerase switching and replication fork management. In summation, we anticipate that the study of the coordinated regulation of DNA replication, DNA damage tolerance and repair, and cell cycle progression in E. coli will provide a valuable framework for understanding these same processes in humans, where the complexity of the events is far greater.