Women with germline mutations of the BRCA1 gene face lifetime risks of 80% for breast cancer and 40% for ovarian cancer. The wildtype allele of BRCA1 is typically lost or inactivated in the tumors that arise in these patients, implying that BRCA1 normally functions as a tumor-suppressor. However, the mechanism by which BRCA1 suppresses tumor formation in normal breast and ovarian tissues is not understood and the amino acid sequence of the BRCA1 gene product provides few clues about its biochemical function. Nevertheless, BRCA1 contains two protein motifs that are well-conserved phylogenetically: an amino-terminal RING domain and a carboxy-terminal BRCT domain. Furthermore, these motifs serve as common sites for BRCA1 missense mutations that predispose women to early-onset breast and ovarian cancer. Together, these data suggest that the RING and BRCT domains are especially important for tumor suppression by BRCA1. To obtain insights into the mechanism of BRCA1-mediated tumor suppression, the applicant sought to identify proteins that associate in vivo with BRCA1. His initial efforts led to the identification of BARD1, a novel protein that resembles BRCA1 in that it possesses an amino-terminal RING domain and a carboxy-terminal BRCT domain. The structural similarities between these polypeptides and their ability to form a stable protein complex in vivo suggest that BARD1 may serve as a regulator and/or effector of BRCA1-mediated tumor suppression. The applicant proposes to test this hypothesis by characterizing the biochemical properties of BARD1 and by evaluating its functional relationship with BRCA1. Accordingly, the Specific Aims of the proposed study are (1) to determine the subcellular localization of BARD1, (2) to define the expression of BARD1 during cell cycle progression, (3) to evaluate the transactivation potential of BARD1 sequences, (4) to examine the DNA-binding activity of BRCA1 and BARD1, (5) to identify proteins that interact with BARD1 in vivo, and (6) to evaluate the effect of BARD1 on BRCA1-mediated tumor suppression.
