The first step in understanding breast cancer is to decipher the exact mode by which cells become cancerous. To accomplish this task, we have initiated a direct biochemical approach to isolate and characterize the role of the proteins that cause breast cancer. One such protein, BRCA1, is shown to be a component of a larger machinery. We reported the isolation of one such engine, termed BRCA1-SWI/SNF, playing a role in transcriptional control. However, our analysis revealed the existence of other BRCA1-containing machines containing the tumor suppressor BARD1. Recently, the BARD1-BRCA1 complex was shown to exhibit a ubiquitin ligase activity associated with polypeptides involved in protein processing. To date, there has been no attempt to purify BARD1-containing complexes. Biochemical purification can yield valuable insights into the polypeptide composition and the functional role of multiprotein complexes. Although genetic approaches have been successful in defining the genes that are mutated in breast cancer, functional understanding of the protein product of these genes requires biochemical studies. We hypothesize that BRCA1 plays a role in the maintenance of genome stability through its interaction with BARD1. Testable predictions of this hypothesis are: (1) Mutations in BRCA1 known to be associated with a predisposition to breast cancer might disrupt the association of either BRCA1 or important components of the BARD1-BRCA1 complex. (2) BRCA1-BARD1 complex prepared from cells carrying BRCA1 mutations might display defects in ubiquination assays. We will use biochemical techniques that have been instrumental in increasing our understanding of the transcription machinery and that have not yet been applied in studies of BARD1 to isolate the BARD1-BRCA1 complex. We intend to identify BRCA1-associated proteins that are altered as the result of mutations in BRCA1 protein.