Saturday, December 21, 2024
12/21/2024
Project Summary RAD51-dependent homologous recombination (HR) is essential for faithful DNA double-strand break (DSB) repair and thus safeguards against genome rearrangements, neoplastic cell transformation, and oncogenesis. During HR initiation, DSBs are processed, yielding ssDNA overhangs that are swiftly coated by Replication Protein A (RPA) to preserve these DNA overhangs and to activate the DNA damage checkpoint. Several “HR mediators” play a key role in RPA-RAD51 exchange to help ensure timely assembly of RAD51-ssDNA nucleoprotein filaments capable of conducting DNA homology search and subsequent strand invasion. RAD51 paralogs (RAD51B, RAD51C, RAD51D, XRCC2, and XRCC3) are HR mediators that assemble into distinct CX3 (RAD51C-XRCC3) and BCDX2 (RAD51BCD-XRCC2) complexes that mediate RAD51 filament assembly. Germline mutations in members of these RAD51 paralog complexes give rise to breast, ovarian and other cancers, and are causative of the cancer-prone syndrome Fanconi anemia. This underscores the crucial roles of RAD51 paralogs in genome maintenance and tumor suppression. Despite their fundamental importance to genome integrity and their involvement in cancer susceptibility, little is known about the mechanisms by which BCDX2 and CX3 serve as mediators of RAD51-dependent HR. The goal of this proposal is to determine the structural and molecular basis by which the CX3 (Aim 1) and BCDX2 (Aim 2) promote RAD51-ssDNA nucleoprotein filament assembly and how their dysregulation leads to cancer. Our studies will also shed light on how higher order complexes of CX3 and BCDX2 with partner proteins, such as PALB2 and HELQ, mediate RPA-RAD51 exchange on ssDNA, confer cellular resistance to DNA damaging agents, and function in the protection of stress and damaged DNA replication forks against nucleolytic attrition. Mechanistic questions regarding the molecular roles of these RAD51 paralog complexes will be answered by our Multi-PI research team through a combination of single particle cryo-EM, single- molecule DNA curtain assays, together with biochemical, biophysical, and cell-based analyses. We expect our multidisciplinary, synergistic approach to yield a deeper understanding of how CX3 and BCDX2 complexes fulfil their crucial function in HR and to furnish actionable information for the development of small molecule cancer therapeutics targeting distinct steps of RAD51-mediated HR pathway.
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