The role of human RAD52 protein in genome stability

人类 RAD52 蛋白在基因组稳定性中的作用

• 批准号: 9904590
• 负责人: Anna L Malkova
• 金额: $ 41.85万
• 依托单位: UNIVERSITY OF IOWA
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-04-01 至 2024-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9904590
• 关键词: Antineoplastic Agents; BRCA1 gene; BRCA2 gene; Biochemical; Biological Assay; Cell Cycle Stage; Cell Death; Cells; Chickens; Complex; DNA; DNA Repair Gene; DNA biosynthesis; DNA replication fork; Dangerousness; Data; Development; Distress; Drug Targeting; Event; Failure; Fiber; Filament; Fluorescence Microscopy; Gatekeeping; Genetic; Genetic Materials; Genome; Genome Stability; Goals; Grant; Human; Lead; Learning; Ligation; Maps; Mediating; Modeling; Motor; Mutation; Neoplastic Cell Transformation; Nucleoproteins; Play; Proteins; RAD52 gene; Recovery; Role; Stress; Stretching; Testing; Time; Tumor Suppressor Proteins; Work; base; bioinformatics tool; brca gene; cell growth; cell killing; foot; genome sequencing; mutant; nuclease; prevent; programs; repaired; replication stress; single molecule; whole genome

The accurate and timely DNA replication program is a prerequisite of a stable genome. This proposed project is built around our discovery that the RAD52 DNA repair protein performs an important and previously unknown function in supporting DNA replication. RAD52 protects replication forks from excessive degradation, which depends on fork regression and on the MRE11 nuclease. Several mechanisms, including a well characterized mechanism ascribed to the activities of SMARCAL1, ZRANB3, BRCA1, BRCA2 and RAD51, protect replication forks stalled by damage or endogenous roadblocks due to the replication stress. Distinctly, the function of RAD52 in fork protection is relevant not only after induction of fork stalling by exogenous stress, but also during an unchallenged cell growth. Our goal here is to develop a comprehensive mechanistic understanding of the RAD52 function at the replication fork. The MRE11-dependent degradation of the replication forks depends on fork regression, i.e. on the conversion of a three-way junction of stalled replication fork into a four-way junction called “chicken foot”. We propose that one or both of the following non-mutually exclusive mechanisms contribute(s) to RAD52 function at the replication forks. In the first, RAD52 may serve as a gatekeeper by preventing regression of stalled, but undamaged forks. In the second, RAD52 may work as a protector of regressed forks either together with, or in parallel to the BRCA1/BRCA2/RAD51 axis. In AIM 1 and AIM 2 we will use cell-based analyses, stretched DNA fibers, proximity-ligation assays and single-molecule total internal reflection fluorescence microscopy (smTIRFM) to test the gatekeeper and the protector mechanisms. By building a comprehensive mechanistic description of the RAD52-fork interaction in the cell and in singulo we will discern whether one or both of these mechanisms are applicable to RAD52 and how RAD52 contributes to replication fork stability. In AIM 3, to characterize the consequences of the RAD52 deficiency, we will combine the cell-based assays, stretched DNA fibers, smTIRFM with the analysis of whole genome sequences by MMBIRFinder, which is a new bioinformatics tool we developed to detect complex mutation events. We will determine the mechanism(s) by which the aberrant recovery of DNA replication is funneled into different genome destabilizing mechanisms in the presence and absence of RAD52. Upon successful completion of the proposed studies we will learn how RAD52 functions at distressed replication forks, how does it contribute to genome stability and how its deficiency leads to genome destabilizing events during replication stress.

准确及时的DNA复制程序是基因组稳定的先决条件。这个提议 该项目是围绕我们的发现而建立的，即 RAD52 DNA 修复蛋白具有重要且 以前未知的支持 DNA 复制的功能。 RAD52 保护复制叉免受 过度降解，这取决于叉回归和 MRE11 核酸酶。一些 机制，包括归因于 SMARCAL1、ZRANB3、 BRCA1、BRCA2 和 RAD51，保护因损伤或内源性障碍而停滞的复制叉 到复制压力。显然，RAD52 在叉子保护中的功能不仅与 外源应激诱导叉失速，而且在未受挑战的细胞生长过程中也能诱导叉失速。 我们的目标是对 RAD52 功能有一个全面的机械理解 复制叉。 复制叉的 MRE11 依赖性退化取决于复制叉回归，即 将停滞的复制叉的三路连接点转换为称为“鸡脚”的四路连接点。 我们建议以下一种或两种非互斥机制有助于 RAD52 在复制叉上发挥作用。首先，RAD52 可以充当看门人，防止回归 停顿但未损坏的货叉。在第二种情况下，RAD52 可以作为回归分叉的保护器 与 BRCA1/BRCA2/RAD51 轴一起或平行。 在 AIM 1 和 AIM 2 中，我们将使用基于细胞的分析、拉伸 DNA 纤维、邻近连接分析和 单分子全内反射荧光显微镜（smTIRFM）来测试看门人和 保护机制。通过构建 RAD52 前叉的全面机械描述 细胞中的相互作用以及单个的相互作用，我们将辨别这些机制中的一种或两种是否适用 RAD52 以及 RAD52 如何促进复制叉稳定性。 在 AIM 3 中，为了描述 RAD52 缺陷的后果，我们将结合基于细胞的 化验、拉伸 DNA 纤维、smTIRFM 以及通过 MMBIRFinder 分析全基因组序列， 这是我们开发的一种新的生物信息学工具，用于检测复杂的突变事件。我们将确定 DNA复制的异常恢复被汇集到不同基因组的机制 RAD52 存在和不存在时的不稳定机制。 成功完成拟议的研究后，我们将了解 RAD52 在不良情况下如何发挥作用 复制叉，它如何促进基因组稳定性以及它的缺陷如何导致基因组 复制压力期间的不稳定事件。