Project Summary/Abstract Homologous recombination, i.e., homology-directed repair (HDR), is a major repair pathway for double-strand breaks (DSBs), including lesions arising during DNA replication. HDR mutants are characterized by genomic instability and sensitivity to DNA damaging agents such as interstrand cross-linking agents like cisplatin and poly(ADP-ribose) polymerase inhibitors, both of which are used in cancer treatment. Several proteins central to the HDR pathway are tumor suppressors, notably the breast and ovarian cancer suppressor BRCA2, which promotes the function of RAD51, the critical protein for homologous strand exchange. RAD51 paralogs are also key HDR proteins and have also been identified both as tumor suppressors and as proteins that affect therapy response. These HDR proteins are essential. Individuals with germline mutations are constitutionally heterozygous, but tumors typically have somatic undergone loss of heterozygosity (LOH), losing the wild-type allele, presumably as an early step in tumor initiation. This proposal has an overarching goal of integrating our understanding how HDR proteins act to maintain genomic stability and cell and tissue homeostasis, how they come to be “lost” in cells, and how their function can be restored. Thus, this broad goal impacts tumor initiation, therapy response, and therapy resistance. It incorporates molecular analysis of HDR protein function, with a particular focus on BRCA2, and delineates how cells respond to HDR protein loss, including how they escape cell death to allow tumor formation. Within this goal is understanding tumor initiation from the standpoint of determining mechanisms of LOH that lead to HDR protein loss, as well as uncovering factors that affect LOH frequencies. While HDR protein loss sensitizes tumors to targeted therapies, HDR function is often restored by secondary mutations, leading to therapy resistance. Understanding which mutations are susceptible to reversion and how therapy impacts reversion is of major interest. Finally, HDR within tissues is also part of this integrated goal, in particular, within the fallopian tube epithelium, which is considered the tissue of origin of high-grade serous ovarian cancers.