The role of human RAD52 protein in genome stability

准确、及时的DNA复制程序是获得稳定基因组的前提。这项建议 该项目是围绕我们的发现而建立的，即RAD52 DNA修复蛋白发挥着重要的 以前未知的在支持DNA复制中的作用。RAD52保护复制分叉不受 过度降解，这取决于分叉回归和Mre11核酸酶。几个 机制，包括归因于SMARCAL1、ZRANB3、 BRCA1、BRCA2和RAD51可保护因损坏或内源性障碍而停滞的复制分叉 与复制压力有关。显然，RAD52在叉子保护中的作用不仅与 外源胁迫诱导叉子失速，但也是在无挑战的细胞生长期间。 我们的目标是从机制上对RAD52的功能有一个全面的理解 复制分叉。 依赖于mre11的复制分叉的退化取决于分叉回归，即 将停滞不前的复制叉子的三向连接转换为称为“鸡爪”的四向连接。 我们建议，以下两种非互斥机制中的一种或两种有助于(S)RAD52 在复制分叉上发挥作用。在第一种情况下，RAD52可以通过防止倒退来充当看门人 停滞不前但没有损坏的叉子。在第二种情况下，RAD52也可以作为退化叉的保护者 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、拉伸的DNA纤维、邻近连接分析和 单分子全内反射荧光显微镜(SmTIRFM)检测门卫和 保护者机制。通过建立对RAD52叉子的全面机械描述 在细胞中的相互作用，我们将辨别这两种机制中的一种或两种都适用 RAD52以及RAD52如何有助于复制分叉的稳定性。 在AIM 3中，为了描述RAD52缺陷的后果，我们将结合基于细胞的 分析，拉伸的DNA纤维，smTIRFM与MMBIRFinder的全基因组序列分析， 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。我们将确定 DNA复制异常恢复进入不同基因组的机制(S) 在RAD52存在和不存在的情况下的不稳定机制。 在成功完成建议的研究后，我们将了解RAD52如何在DISRISTED工作 复制分叉，它如何有助于基因组稳定以及它的缺陷如何导致基因组 复制应激期间的不稳定事件。

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.