Replication checkpoint activation and silencing

描述（由申请方提供）：细胞周期S期期间的DNA复制过程不断受到复制模板上存在受损DNA的挑战。染色体中的碱基损伤可导致DNA聚合酶停滞，并且如果停滞的聚合酶未被解决，则复制叉将崩溃，并且染色体将断裂。因此，折叠的复制叉是对维持基因组稳定性的严重威胁，并且被认为是产生允许正常细胞变成癌细胞的遗传不稳定性的主要事件。在本项目中，我们将重点关注允许细胞在S期耐受DNA损伤的两条重要途径，即ATM和Rad 3相关（ATR）依赖性复制检查点以及DNA聚合酶eta依赖性跨损伤合成（TLS）损伤旁路途径。我的实验室最近的结果表明，这两个途径在DNA损伤反应过程中相互作用，特别是，pol eta可以覆盖DNA损伤对ATR的激活。在这个项目中，我们将通过研究关键的ATR激活器TopBP 1，来关注ATR是如何被停滞的分叉激活的。我们已经发现TopBP1感觉到停滞的分叉，并且它招募DNA聚合酶α（pol 1）和911复合物到停滞的分叉。这两个因子被TopBP1募集是ATR激活所必需的。在目标1中，我们将研究TopBP 1感知停滞叉的分子机制，在目标2中，我们将探索它如何招募pol 1和911的生化机制。在目标3中，我们将研究pol eta如何超越DNA损伤的ATR反应，在目标4中，我们将研究一种新的，基于蛋白水解的机制，在DNA损伤反应过程中调节pol eta功能。如果这些目标得以实现，那么我们将对ATR激活和pol eta调节的分子机制有更深入的了解。重要的是，我们还将增加对ATR和pol eta途径如何相互作用的理解，这将使细胞如何管理复制应激的观点更加综合。 公共卫生相关性：DNA损伤是染色体复制的严重障碍，染色体复制是细胞分裂过程的基本组成部分。当染色体复制过程中遇到DNA损伤时，它会使负责复制遗传物质的DNA聚合酶停止。这种停滞可能对基因组的稳定性产生严重后果，因为停滞的聚合酶未得到解决可能导致复制过程崩溃，染色体断裂。断裂染色体的修复可能是不完美的，并且因此可能导致已知导致癌症的染色体易位。在这个建议中，我们专注于两个细胞途径，帮助细胞处理停滞的聚合酶。一个是信号通路，我们将研究这个信号通路如何识别停滞的聚合酶，以及它如何被它们激活。另一种途径涉及一种专门的DNA聚合酶，即使在DNA受损时也可以复制DNA。我们将研究这种聚合酶的调节，以及这种聚合酶影响来自停滞复制的信号的能力。这些研究将帮助我们了解细胞如何管理停滞的复制，并可能为更新，更有效的抗癌药物奠定基础。

DESCRIPTION (provided by applicant): The process of DNA replication during S phase of the cell cycle is constantly challenged by the presence of damaged DNA on the replication template. Base lesions in chromosomes can cause DNA polymerase stalling, and if the stalled polymerase is not resolved than the replication fork will collapse, and the chromosome will be broken. Collapsed replication forks are, therefore, a serious threat to the maintenance of genome stability, and are thought to be a primary event in generating the genetic instability that allows normal cells to become cancer cells. In this project, we will focus on two important pathways that allow cells to tolerate DNA damage during S phase, the ATM and Rad3 related (ATR)- dependent replication checkpoint, and the DNA polymerase eta-dependent trans- lesion synthesis (TLS) damage bypass pathway. Recent results from my laboratory have revealed that these two pathways interact during a DNA damage response and, in particular, that pol eta can override the activation of ATR by DNA damage. In this project, we will focus on how ATR is activated by stalled forks, by studying the critical ATR activator TopBP1. We have found that TopBP1 senses the stalled fork, and that it recruits DNA polymerase alpha (pol 1) and the 911 complex to the stalled fork. Recruitment of these two factors by TopBP1 is required for ATR activation. In Aim 1, we will investigate the molecular mechanism whereby TopBP1 senses stalled forks, and in Aim 2 we will probe the biochemical mechanism for how it then recruits pol 1 and 911. In Aim 3, we will investigate how pol eta overrides the ATR response to DNA damage, and in Aim 4 we will investigate a novel, proteolytic-based mechanism that regulates pol eta function during the DNA damage response. If these goals are met, then we will have achieved a greater understanding of the molecular mechanisms involved in ATR activation, and in pol eta regulation. Importantly, we will have also increased out understanding of how the ATR and pol eta pathways interact, and this will allow for a more integrated view of how cells manage replication stress to emerge. PUBLIC HEALTH RELEVANCE: DNA damage is a serious impediment to chromosome replication, a fundamental component of the process of cell division. When DNA damage is encountered during chromosome replication, it will stall the DNA polymerases that are responsible for duplication of the genetic material. This stalling can have severe consequences for the stability of the genome, as a stalled polymerase that is left unresolved can cause the replication process to collapse, and the chromosome to break. The repair of broken chromosomes can be imperfect, and can than thereby result in the chromosome translocations that are known to cause cancer. In this proposal, we focus on two cellular pathways that help cells deal with stalled polymerases. One is a signaling pathway, and we will study how this signaling pathway recognizes stalled polymerases and, how it is activated by them. The other pathway involves a specialized DNA polymerase that can replicate DNA even when it is damaged. We will study the regulation of this polymerase, and the ability of this polymerase to influence signaling that is derived from stalled replication. These studies will help us understand how cells manage stalled replication, and could form the basis for newer and more effective anti-cancer drugs.