PROJECT SUMMARY PARP inhibitors (PARPi) hold tremendous therapeutic potential because of their selectivity for cells lacking functional BRCA1, BRCA2, and other homology-directed repair (HDR) genes. However, as with other targeted therapies, resistance to PARPi frequently arises, underscoring the unmet need to elucidate how PARPi cause cell death in BRCA mutant but not normal cells. Individual PARPi may act through distinct mechanisms, either by “trapping” PARP-DNA complexes, or by inhibiting repair of single-stranded (ssDNA) nicks that are subsequently converted to double-stranded breaks (DSBs). Moreover, patients may exhibit differential drug sensitivity depending on the specific causative BRCA gene mutation. Defining this fundamental landscape will be critical to better predict responders/non-responders as well as the durability of patient response to PARPi. Historically, a detailed, mechanistic study of how mutations in BRCA2 influence genome integrity has been hampered by the immense challenge of manipulating and purifying this large protein. Recently, we have overcome these challenges, allowing us to leverage a combination of in vitro biochemical assays and cellular assays to pinpoint how individual pathogenic or targeted mutations influence specific functionalities including: DNA binding, replication fork protection, RAD51 nucleoprotein filament formation, and RAD51-mediated DNA strand invasion. In addition to applying these techniques to interrogate the explicit biochemical function(s) compromised by pathogenic BRCA2 mutations, we will assess sensitivity to PARPi with strong, intermediate, or weak trapping activity (e.g. Talazoparib, Olaparib, and Veliparib, respectively). Lastly, we will investigate the function(s) reconstituted by “reversion” mutations identified in patients with PARPi-resistant tumors, which may independently identify functional attributes necessary for PARPi sensitivity. Our long-term goal is to unveil the molecular consequences of PARPi treatment that necessitate processing by BRCA2, RAD51, and other HDR proteins. Our central hypothesis is that by elucidating how BRCA2 and RAD51 mechanistically overcome PARPi-mediated toxicity, we will provide the necessary framework to understand how PARPi resistance can develop in patients. Our hypothesis is based on compelling preliminary data illustrating the specific functions of BRCA2 and RAD51 in response to PARPi. Thus, our rationale, to reveal the mechanism(s) that underlie PARPi-mediated toxicity, will vertically advance knowledge surrounding the HDR response to PARPi, and ultimately, improve clinical management of BRCA patients. In aim 1, we will utilize patient derived BRCA2 reversion alleles in our isogenic human cell models to interrogate what specific function(s) have been “reactivated” to promote resistance to PARPi. In aim 2, we will determine how BRCA2 and RAD51 catalyze the removal or bypass of PARPi trapped lesions using purified proteins and relevant model DNA sub...