Abstract IA024: Mechanistic insights into how RADX regulates RAD51 nucleoprotein filaments to maintain genome stability and control replication stress responses

Abstract

Abstract RAD51 nucleoprotein filaments are central to maintaining genome stability, governing crucial processes like homology-directed double-strand break repair, replication fork reversal, and shielding replication forks from nucleases. The precise regulation of RAD51 filament formation and stability is critical for these functions, which suppress tumorigenesis and determine cellular responses to common cancer therapies. RADX is a pivotal regulator of RAD51 in the context of DNA replication, impacting replication fork reversal and fork stabilization. After identifying RADX as an RPA-related RAD51 regulator, we have worked to understand how it acts, thereby elucidating its role in genome stability and its influence on cancer cell responses to PARP inhibitors and chemotherapies. Genetically, RADX exhibits a dual role, capable of either inhibiting or promoting replication fork reversal based on the levels of replication stress. Biochemical studies show that RADX has inhibitory effects on RAD51 strand exchange and D-loop formation activities, achieved through direct binding to single-strand DNA and RAD51, along with the stimulation of RAD51 ATP hydrolysis. These activities collectively destabilize RAD51 nucleofilaments, opposing the stabilizing effects of BRCA2. Cells lacking RADX regulatory functions exhibit replication defects, DNA damage accumulation, reduced growth, and heightened sensitivity to DNA damage and replication stress. Structural analyses, including cryo-electron microscopy and mass photometry, revealed how RADX binds ssDNA and show it exists in multiple oligomeric states with a preference for trimers when bound to single-stranded DNA. Negative stain electron microscopy imaging supports a model wherein RADX functions by capping and restricting the growing ends of RAD51 filaments. In summary, our findings provide a comprehensive understanding of the regulatory mechanisms governing RAD51 nucleofilament dynamics by RADX, emphasizing its crucial role in coordinating replication fork stability and genome integrity. This knowledge not only contributes to the fundamental understanding of cellular processes but also offers insights into potential therapeutic interventions targeting RAD51-controlled pathways. Citation Format: Madison Adolph, Swati Balakrishnan, Walter Chazin, David Cortez. Mechanistic insights into how RADX regulates RAD51 nucleoprotein filaments to maintain genome stability and control replication stress responses [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Damage Repair: From Basic Science to Future Clinical Application; 2024 Jan 9-11; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2024;84(1 Suppl):Abstract nr IA024.