Replication checkpoint activation and silencing

复制检查点激活和静默

• 批准号: 7900280
• 负责人: MATTHEW MICHAEL
• 金额: $ 9.44万
• 依托单位: HARVARD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-08-13 至 2010-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7900280
• 关键词: ATM Signaling Pathway; ATM activation; ATR protein kinase; Antineoplastic Agents; Attenuated; Biochemical; Biochemical Genetics; Biological Assay; Bypass; Caenorhabditis elegans; Cancer Etiology; Cell Cycle; Cell Cycle Checkpoint; Cell Division Process; Cells; Chromosomal Breaks; Chromosomal translocation; Chromosome Breakage; Chromosomes; Complex; DNA; DNA Damage; DNA Polymerase I; DNA biosynthesis; DNA-Directed DNA Polymerase; Embryo; Event; Genetic; Genetic Materials; Genetic Screening; Genome Stability; Goals; Human; Laboratories; Lead; Left; Lesion; Maintenance; Mediating; Molecular; Normal Cell; Orthologous Gene; Pathway interactions; Phase; Physiological; Play; Polymerase; Process; Protein Kinase; Proteins; Rad30 protein; Recruitment Activity; Regulation; Replication-Associated Process; Role; S Phase; Signal Pathway; Signal Transduction; Site; Stress; System; Xenopus; base; cancer cell; chromosome replication; egg; genetic analysis; meetings; novel; public health relevance; repaired; response; ubiquitin ligase; ubiquitin-protein ligase

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.

描述（由申请人提供）：细胞周期s期间的DNA复制过程不断受到复制模板上受损的DNA的挑战。染色体中的碱病变会引起DNA聚合酶失速，并且如果无法分辨出停滞的聚合酶，则比复制叉将塌陷，并且染色体将损坏。因此，折叠的复制叉是对维持基因组稳定性的严重威胁，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。在这个项目中，我们将重点关注两种重要的途径，这些途径使细胞在S期间耐受DNA损伤，ATM和RAD3相关（ATR） - 依赖性复制检查点以及DNA聚合酶ETA依赖性trans-依赖性trans循环合成（TLS）损伤旁路途径。我的实验室的最新结果表明，这两种途径在DNA损伤响应中相互作用，尤其是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 Activator topBP1来关注ATR如何被失速的叉子激活。我们发现TOPBP1感应失速的叉子，并且它募集了DNA聚合酶α（POL 1）和911复合物，并将其募集到失速的叉子上。 ATR激活需要通过TOPBP1募集这两个因素。 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.如果实现了这些目标，那么我们将对ATR激活和POL ETA调节中涉及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，这将使人们对细胞如何管理复制应力的出现更加整合。 公共卫生相关性：DNA损伤是对染色体复制的严重障碍，这是细胞分裂过程的基本组成部分。当染色体复制期间遇到DNA损伤时，它将陷入导致遗传物质重复的DNA聚合酶。这种停滞可能会对基因组的稳定性产生严重的后果，因为静止不动的聚合酶可能导致复制过程塌陷，并且染色体破裂。破裂的染色体的修复可能是不完美的，因此可以导致已知会导致癌症的染色体易位。在此提案中，我们专注于两个细胞途径，以帮助细胞处理停滞的聚合酶。一个是一个信号通路，我们将研究该信号通路如何识别停滞的聚合酶以及如何被它们激活。另一种途径涉及一种专门的DNA聚合酶，即使DNA损坏也可以复制DNA。我们将研究该聚合酶的调节，以及该聚合酶影响从停滞的复制中产生的信号传导的能力。这些研究将有助于我们了解细胞如何管理停滞的复制，并可能构成更新，更有效的抗癌药物的基础。