BRCA1 and BRCA2 have established functions in homologous recombination (HR), particularly in the repair of replication-associated DNA breaks by sister chromatid recombination (SCR). Many rare BRCA1 missense mutant alleles, termed "variants of uncertain significance" (VUS), are difficult to classify as pathogenic or benign, due o their scarcity in the human population. Therefore, for a woman who carries a germ line BRCA1 VUS allele, the risk of developing breast or ovarian cancer is unknown. A major goal of this proposal is to study a large panel of ~150 BRCA1 variants, including ~60 BRCA1 VUS alleles in HR/SCR, using highly innovative SCR reporters that are unique to the Scully lab. Our work will reveal fundamental mechanisms of action of BRCA1 in HR. Further, it will advance human health by helping to predict cancer risk for individuals who carry a BRCA1 VUS allele in the germ line and, hence, make informed decisions regarding prophylactic measures. AIM 1: Perform a genetic analysis of a large panel of BRCA1 variants in homologous recombination and LTGC suppression. We developed novel flow cytometric reporters of SCR, triggered by a site-specific chromosomal double strand break (DSB). We discovered that, in addition to its known function in "overall HR", BRCA1 also specifically suppresses aberrant "long tract" gene conversion (LTGC) between sister chromatids. Each of these defects is corrected by wild type human (h)BRCA1 but not by known pathogenic missense hBRCA1 alleles. In this Aim we will extend our analysis to include a large number of BRCA1 variants, including VUS alleles, with the goal of developing a robust predictor of the pathogenicity of specific BRCA1 VUS alleles. This Aim will include analysis of BRCA1 SCR functions in normal development and during breast tumorigenesis in a mouse model. AIM 2: Define mechanisms by which BRCA1 controls hr at Tus/ter-arrested replication forks. A long-standing hypothesis proposes that BRCA1 controls HR at stalled replication forks. This has been difficult to test, due to the dearth of tractable tools for stalling the mammalian replication fork at a defined chromosomal locus. We recently solved this problem by adapting the Escherichia coli Tus/ter replication fork arrest complex for use in mammalian cells. We discovered that BRCA1 controls HR at Tus/ter-stalled replication forks, but that HR in this context is regulated differently from HR induced by a "generic" restriction endonuclease-induced chromosomal DSB. Remarkably, loss of wtBRCA1 causes an increase in the absolute frequency of LTGC events at Tus/ter-stalled replication forks. In this proposal, we will analyze how BRCA1 controls HR at stalled forks and will attempt to identify the specific mechanisms by which BRCA1 suppresses LTGC at Tus/ter-stalled mammalian chromosomal replication forks. PUBLIC HEALTH RELEVANCE: If a woman inherits an error ("mutation") in the BRCA1 gene, she will have a ~10-fold increased risk of breast and ovarian cancer throughout her life. However, some BRCA1 mutations occur too rarely in the population to allow the associated risk of breast or ovarian cancer to be determined. Our goal here is to use new tools that the Scully lab has developed for studying the function of BRCA1 to help identify which of the rare BRCA1 variants are benign and which confer increased risk of cancer.