PROJECT SUMMARY Error-free DNA repair initiated at the sites of replication fork stalling is critical for the prevention of genomic instability in cycling cells. Defects in stalled fork repair have been directly implicated in cancer and other human diseases, notably in hereditary breast and ovarian cancer (HBOC), in light of the involvement of BRCA1 and BRCA2 in repair of stalled replication forks. Our work previously established roles for BRCA1 and BRCA2 in regulating HR at both double strand breaks (DSBs) and in stalled fork repair. We have developed innovative tools for quantifying homologous recombination (HR) and other repair outcomes at stalled mammalian replication forks and, more recently, at broken replication forks. Major goals of this proposal are to define how BRCA1 contributes to the repair of stalled or broken forks. We anticipate that its roles in response to these two distinct lesions may be quite different. We have developed an array of cutting-edge tools to support this study, including unique, sophisticated HR reporters that can distinguish between error-free “short tract” HR and error- prone “long tract” HR—a replicative response likely analogous to break-induced replication as described in yeast. One unusual aberrant replicative response that we observe at stalled forks specifically in BRCA1 mutant cells is the formation of ~10 kb non-homologous tandem duplications (TDs). Remarkably, these highly specific forms of structural variation are also abundant in the BRCA1-linked breast and ovarian cancer genome. Another major goal of this proposal is to define more fully the genetic regulation of TD formation at stalled forks. Success in this project will reveal the mechanisms that regulate mammalian stalled (or broken) fork repair and their relationship to HBOC predisposition in unprecedented detail. This work may also identify new molecular targets for therapy of breast and ovarian cancer.