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: 10361559
• 负责人: Anna L Malkova
• 金额: $ 40万
• 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/10361559
• 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前提。这一拟议 该项目是围绕我们的发现，即RAD 52 DNA修复蛋白执行一个重要的， 支持DNA复制的功能。RAD 52保护复制分叉， 过度降解，这取决于叉回归和MRE 11核酸酶。几 机制，包括归因于SMARCAL 1，ZRANB 3， BRCA 1、BRCA 2和RAD 51，保护因损坏或内源性障碍而停滞的复制叉， 复制压力。显然，RAD 52在叉保护中的功能不仅在 通过外源性应激诱导叉停滞，而且在未受攻击的细胞生长期间。 我们的目标是对RAD 52功能有一个全面的机械理解， 复制分叉。 复制分叉的MRE 11依赖性降级取决于分叉回归，即 将停滞的复制叉的三向连接转换为称为“鸡脚”的四向连接。 我们建议，以下非互斥机制中的一个或两个有助于RAD 52 在复制分叉处运行。在第一种情况下，RAD 52可以通过防止回归来充当看门人 但没有损坏的叉子在第二种情况下，RAD 52可以作为回归分叉的保护器， 与BRCA 1/BRCA 2/RAD 51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，邻近连接测定和 单分子全内反射荧光显微镜（smTIRFM）测试看门人， 保护机制。通过建立一个全面的机械描述的RAD 52-叉 在细胞和singulo中的相互作用，我们将辨别这些机制中的一个或两个是否适用。 到RAD 52以及RAD 52如何对复制分叉稳定性做出贡献。 在AIM 3中，为了表征RAD 52缺陷的后果，我们将联合收割机结合基于细胞的 分析，拉伸DNA纤维，smTIRFM与全基因组序列的MMBIRTM分析， 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。康贝特人将以 DNA复制的异常恢复通过漏斗进入不同基因组的机制 在存在和不存在RAD 52的情况下的不稳定机制。 在成功完成拟议的研究后，我们将了解RAD 52如何在困境中发挥作用。 复制叉，它如何有助于基因组的稳定性，以及它的缺陷如何导致基因组 在复制应激期间的不稳定事件。