Contact PD/PI: Greenberg, Roger A. Summary Faithful execution of homologous recombination DNA repair is essential for genome integrity and is a critical determinant of cancer etiology and therapeutic response. This relationship is prominently exemplified by hereditary breast and ovarian cancer, which arises as a consequence of germline mutations to BRCA1 and a network of genes encoding its interacting partners. Dysfunction within this BRCA network produces hypersensitivity to poly(ADP)ribose polymerase inhibitors, which are clinically approved agents that have efficacy in the setting of BRCA mutation and homologous recombination deficiency in general. Resistance frequently occurs to these agents as a consequence of restored homologous recombination DNA repair. It is thus essential to understand how this occurs at a fundamental mechanistic level. We discovered that BRCA1 is targeted to lysine63-linked ubiquitin chromatin regions aligning DNA double-strand breaks and damaged replication forks. This localization requires BRCA1 interaction with the A-complex. This dimeric complex of five proteins includes a ubiquitin binding protein, RAP80, and a deubiquitinating enzyme, BRCC36. RAP80 and BRCC36 are specific for binding lysine63-linked ubiquitin and its hydrolysis. Deficiency of the A-complex reduces BRCA1 DNA damage localization and causes excessive end resection at replication fork damage. Interestingly, loss of the A-complex confers resistance to PARP inhibitors in genetic backgrounds where end resection is diminished. How the BRCA1-A-complex links chromatin recognition to control of end resection and therapeutic response is not currently understood. This proposal will integrate in vivo, cellular, and structure guided biochemical approaches to understand how the A-complex links DNA double-strand break ubiquitin recognition on nucleosomes to homologous recombination. To achieve these goals, we develop novel methodologies that enable us to identify the entire DNA damage response proteome on chromatin and how this affects nucleosome modifications and stability. These studies will yield fundamental advances to the understanding of genome integrity control and clarify new strategies to target underlying vulnerabilities in a broad range of malignancies. ! !