Replication checkpoint activation and silencing

描述（由申请人提供）：细胞周期S期的DNA复制过程不断受到复制模板上受损DNA的挑战。染色体上的碱基损伤会导致DNA聚合酶停滞，如果停滞的聚合酶得不到解决，复制叉就会崩溃，染色体就会断裂。因此，崩溃的复制分叉是对维持基因组稳定性的严重威胁，并且被认为是产生遗传不稳定性的主要事件，使正常细胞成为癌细胞。在本项目中，我们将重点关注细胞在S期耐受DNA损伤的两个重要途径，即ATM和Rad3相关（ATR）依赖的复制检查点，以及DNA聚合酶依赖的trans-病变合成（TLS）损伤旁路途径。我的实验室最近的研究结果表明，这两种途径在DNA损伤反应中相互作用，特别是pol - eta可以通过DNA损伤覆盖ATR的激活。在这个项目中，我们将通过研究关键的ATR激活剂TopBP1，重点研究ATR是如何被熄火叉激活的。我们发现TopBP1感知到停滞的分叉，并将DNA聚合酶α (pol 1)和911复合体招募到停滞的分叉上。ATR激活需要TopBP1募集这两个因子。在Aim 1中，我们将研究TopBP1感知停滞分叉的分子机制，在Aim 2中，我们将探讨它如何招募pol 1和911的生化机制。在Aim 3中，我们将研究pol eta如何覆盖ATR对DNA损伤的反应，在Aim 4中，我们将研究一种新的基于蛋白水解的机制，该机制在DNA损伤反应中调节pol eta的功能。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，我们也将增加我们对ATR和pol - eta途径如何相互作用的理解，这将允许对细胞如何管理复制压力有一个更综合的看法。

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.