Sunday, May 11, 2025
5/11/2025
The role of human RAD52 protein in genome stability - 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.
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