My laboratory focuses on the functional analysis of the human breast cancer susceptibility genes, BRCA1 and BRCA2. Breast cancer is the most frequently diagnosed cancer in women in the United States. It has been estimated that about 178,480 new cases of invasive breast cancer were diagnosed and more than 40,400 individuals died from this disease in 2007. Among the various factors responsible for the development of this cancer, a family history of the disease seems to play a major role. Mutations in BRCA1 and BRCA2 are linked to increased risk of early onset familial breast and ovarian cancers. Individuals with mutations in either of these genes are also at risk for developing cancer in other organs as well. The penetrance of the disease in BRCA1 and BRCA2 mutation carriers has been estimated to be 35-80%. In an effort to reduce the mortality from breast cancer through prevention and early diagnosis, BRCA1 and BRCA2mutation carriers are encouraged to undergo intensive screening and, in some cases, prophylactic surgery or chemoprevention. Sequencing based genetic tests are available to identify BRCA1 and BRCA2 mutation carriers. Currently, association analyses in families are used to determine whether a mutation poses a risk. One of the main aims of this project is to understand how deleterious mutations in BRCA1 and BRCA2&lt;/ result in tumor development. By generating such mutations in mice, we hope to improve our undertstanding of the role of BRCA1 and BRCA2 as tumor suppressor. We are generating humanized mouse models using human BRCA1 and BRCA2 present in bacterial artificial chromosomes (BAC) . BRCA1 or BRCA2 mutations are generated in the human BACs and the phenotype is analyzed in transgenic mice that are homozygous null mutants for the endogenous gene. These humanized mouse models provide an experimentally tractable system to generate mutations identified in humans and to analyze the mechanism by which they cause tumorigenesis. Functional analysis of BRCA1 and BRCA2 variants will also help us uncover any novel functions of these proteins. For example, analysis of a small 29-amino acid deletion in an evolutionarily conserved BRCA2 domain has revealed a role in alkyl-DNA repair. Embryonic fibroblasts expressing this mutant allele are sensitive to agents that alkylate guanine bases in the DNA. Repair of alkylated guanine primarily involves removal of the alkyl group by the O6-methylguanine-methyl transferase (MGMT) enzyme. This mutant has enabled us to uncover a role for BRCA2 in the repair of O6-mG adducts in addition to its function in RAD51-mediated DNA repair activity. We have also shown that BRCA2 associates with MGMT and the two proteins undergo degradation after alkylation. We have demonstrated that O6-benzylguanine (O6BG), a non-toxic inhibitor of MGMT, can also induce BRCA2 degradation. Because BRCA2 is a viable target for cancer therapy, our observation that O6BG induces degradation of BRCA2 may have significant clinical implications. We recently generated humanized mouse models expressing BRCA1 variants: M1652I, C61G, A1708E, R1699Q. With the exception of M1652I, which is a neutral variants, all others resulted in embryonic lethality, suggesting that these variants are deleterious. We are now examining the effect of this variants on mammary tumorigenesis by crossing the transgenic mice to Brca1 conditional knockout mice.