Molecular modulator of RAD51 in maintaining genome stability

Genome instability is a hallmark of cancer progression, often exacerbated by replication stress that stalls and disrupts DNA replication, threatening cell survival. Cells use intricate and tightly regulated mechanisms to prevent improper degradation of stalled replication forks by nucleases, a process critical for preserving the genome integrity. Understanding these protective mechanisms is important for advancing cancer therapies, particularly in the context of BRCAness and PARPi resistance. Although there has been significant research on fork protection, the regulatory mechanisms that control nuclease activity at stalled forks remain poorly understood. The primary goal of this project is to elucidate the molecular mechanisms of fork protection. While traditional models have emphasized the role of BRCA2 in recruiting RAD51 to protect stalled replication forks, recent evidence points to alternative pathways, including the involvement of the CST complex. This highlights a significant gap in our understanding of the extensive regulatory mechanisms that ensure fork stability and their implications in cancer therapy resistance. We hypothesize that two parallel RAD51 recruiting mechanisms co- exist in cells, and that the calcium-sensing signaling pathway regulates fork protection. In Aim 1, we will use cryo-EM to capture the RAD51/CST-DNA complex structure at high resolution and examine the functional significance of RAD51/CST interaction in fork protection. In Aim 2, we will apply genomic mapping and functional assays to delineate the roles of BRCA2 and CST in RAD51 recruitment and their potential compensatory relationship in fork protection. In Aim 3, we will investigate how the newly identified replication stress response signaling pathway regulates nuclease processing of stalled forks by examining the role of the phosphorylation of MRE11 in inhibiting aberrant fork processing, potentially identifying new avenues for sensitizing cancer cells to treatment. This application offers multiple conceptual and technical innovations by blending cutting-edge genomics, structural, biochemical, and cell biology techniques to test novel hypotheses. Findings are expected to reshape our fundamental understanding of replication fork protection mechanisms and genome stability.