Project Summary The Fanconi Anemia (FA) pathway forms a complex DNA repair network that can mediate tumor aggressiveness and therapy-resistance when dysregulated in cancers. Although FA proteins have been shown to support cancer survival, the poor characterization of FA pathway biochemical activities that contribute to this phenotype has made therapeutic targeting of these proteins very challenging. Characterization of novel enzymatic roles carried out by FA components could serve as valuable clues into how certain FA proteins are able to promote cancer radiotherapy resistance and would provide identifiable targets for sensitization of cancer cells to traditional treatments. Specifically, FA proteins FANCA, FANCG, and FAAP20, which form a sub-complex (AG20), and FANCA-interacting protein BRCA1 may provide critical care of the unstable cancer genome through FANCA’s intrinsic DNA/RNA binding and newly discovered strand annealing/exchange activities. These activities could contribute to DNA double-strand break repair, which is capable of conferring survival advantages to radiotherapy-treated cancer cells. The goal of this proposal is to further determine additional DNA and RNA processing activities of BRCA1 and the AG20 sub-complex, and how these activities are utilized in a cellular context to preserve the genome. The aims of this project are: 1) identify biochemical activities of BRCA1 and the AG20 sub-complex related to nucleic acid processing events: defining the range of activities AG20 and FANCA/BRCA1 are able to perform using DNA/RNA substrates will be possible using highly active recombinant protein that our lab is well-equipped to generate; 2) determine the chromatin landscape that facilitates FANCA/BRCA1 and AG20-mediated repair: FANCA’s cellular role in DSBs across different chromatin contexts can be analyzed in fine detail through ChIP-Seq analysis of U2OS-DiVA cells present in our lab, and live cell imaging of FANCA recruitment to site-specific DNA damage. By accomplishing these goals, we expect to be able to delineate a working model for the participation of repair factors BRCA1 and AG20 in subsets of DNA damage repair that supports cancer survival and therapy-resistance.