The second breast cancer susceptibility gene, BRCA2, was recently isolated. Breast cancer is highly penetrant in individuals carrying germ-line mutations in this gene. Like BRCA1, it has a large open reading frame that predicts the synthesis of a protein of completely unknown function. Understanding the normal function of BRCA2 and how the loss of this function predisposes towards breast cancer will require a multi-disciplinary approach. This proposal will continue our initial characterization of BRCA2 towards this end. Four aims are described: 1) Reagents for the study of BRCA2 will be developed which include monoclonal antibodies raised against recombinant fragments of the protein, a full length wild-type cDNA and cDNAs with commonly occurring mutations. Initial characterization of the protein will be performed with these reagents. 2) Expression of BRCA2 will be examined in retrospective cohorts of frozen breast and ovarian cancers and compared to normal specimens from these same organs. Altered expression or subcellular location will be noted and compared to established clinico- pathologic parameters. Methylation status of a CpG island in the promoter will be measured and correlated with gene expression. 3) Since BRCA2 expression is regulated with the cell cycle with a dramatic increase observed at the G1/S boundary, its role in processes mediated at this time will be examined. These include apoptosis, differentiation, and response to DNA damaging agents. 4) The BRCA2 promoter responsible for cell cycle regulation will be isolated and the cis and trans acting elements defined. The initial focus will be on the E2F family of transcription factors which regulate other genes with similar cell cycle profile.