According to the American Cancer Society, several million women in the United States have been diagnosed or are undergoing therapy for breast cancer. Breast cancer is a major source of morbidity and mortality both in the United States and throughout the world. Families harboring mutations in BRCA2 are at an especially high risk for developing breast and/or ovarian cancer, as incidence of the disease reaches between 80% and 90% by the age of 70 (compared to 8% in the general population). Understanding the function of BRCA2 and why mutations in this gene predispose affected individuals to such a tissue-specific cancer has the potential to provide new therapeutic strategies for these patients.

Although it is now fully appreciated that mutations in BRCA2 cause cancers, the precise biological function of BRCA2 remains unknown. The importance of BRCA2 in breast cancer is underscored by the fact that mutation of this single gene almost invariably leads to breast cancer in an afflicted individual. A role for BRCA2 in DNA repair has been proposed due to its interaction with a key protein involved in homologous recombination, Rad51. Defects in recombination and other DNA repair pathways have been associated with a wide range of cancer predispositions. Furthermore, previous molecular and cellular analyses have established a connection between BRCA2 and the repair of broken DNA by recombination.

Recombinational DNA repair is a fundamental biological process necessary for the maintenance of chromosomal integrity. The research in this proposal will focus on specific molecular events essential for chromosomal maintenance by BRCA2 and recombinational DNA repair. There is accumulating evidence suggesting that the altered variants of BRCA2 protein result in a mutant protein that fails to interact with another protein, Rad51, which is central to recombinational DNA repair. This loss of the molecular function gives rise to defective recombinational DNA repair, chromosomal instability, and predisposition to cancers of the breast, ovary, and certain epithelial tissues, as well as to hematological malignancies, bone marrow failure, and developmental abnormalities in Fanconi's anemia.

We propose to use a novel single-molecule approach to define how BRCA2, and its constituent parts, regulates the assembly of Rad51 on DNA, using purified human Rad51 and BRCA2 proteins. We plan to use optical methods to trap single molecules of DNA and then, literally, watch the formation of a complex between Rad51 protein, BRCA2, and the trapped DNA. We will explore the effect of BRCA2 on the ability of Rad51 protein to assemble into a filament on this DNA. We expect that our experiments will reveal new information about how these proteins cooperate to repair DNA. Furthermore, they should also uncover the molecular defects that underlie the cancer-causing mutations in BRCA2 protein. These findings should contribute valuable information needed to develop therapeutic strategies that might help overcome the defects in the DNA repair process.