| Abstract | BRCA1 and BARD1 are proteins involved in the repression of genes associated with increased risk for breast and ovarian cancers. This is accomplished through ubiquitination of H2A and subsequent changes in chromatin compaction. BRCA1 and BARD1 form an E3 ligase (BCBD complex), and mutations affecting the enzymatic functions of this complex can predispose women to these cancers. The model organism C. elegans contains orthologs of these proteins, BRC-1 and BRD-1, which makes it a useful organism for studies of protein function; however, little is known about the mechanism of ubiquitination in C. elegans as compared to humans. This project used nucleosome assays to provide more insight on the ubiquitination of H2A by the BCBD complex in C. elegans. The objectives of this project included characterizing the interaction of the BCBD complex with H2A and identifying a specific lysine target in C. elegans. The conserved lysine residues (potential targets) were mutated out of H2A and nucleosome assays were performed to identify potential reductions in ubiquitination activity. The H2A nucleosome assays showed that the mutations of conserved lysines in the H2A N-terminus and C-terminus in C. elegans did not significantly reduce ubiquitination activity, and a definitive target could not be identified through these methods. Further studies are needed to determine if C. elegans has any preferential lysine targets at a non-conserved residue or if it is truly nonspecific in its activity. Currently, mass spectrometry analysis is being performed as a complementary method to attempt to pinpoint the location of lysine ubiquitination. |
