UC Davis

Sexual reproduction relies on successful completion of meiosis, a specialized cell division that produces haploid eggs and sperm; errors in meiosis lead to aneuploid gametes and have devastating consequences on progeny viability. To ensure each gamete receives the correct complement of chromosomes, crossovers are established between each chromosome pair, which along with microtubule attachment, allow for biorientation of the homologous chromosomes across the cell division plane. Many meiotic DNA double strand breaks (DSBs) are purposely introduced in early meiotic prophase to make sure each chromosome pair will receive at least one crossover. The repair of these DSBs by different DNA repair pathways, with homologous recombination (HR) being the major one utilized during meiosis, results in both crossover and non-crossover products. Despite extensive research to understand meiosis, the specific mechanisms for how different repair pathways are utilized for efficient DNA repair, how the position and numbers of crossovers are determined, and how the entire process is orchestrated by different protein players remain elusive. BRCA1 and its binding partner BARD1 are tumor suppressors that play critical roles in maintaining genome integrity by promoting HR in somatic cells. The roles of BRCA1 and BARD1 in meiotic DSB repair, however, are largely unexplored. This is partially due to the two genes being essential in mammals, and null mutations of either gene results in embryonic lethality in mouse models. Further, BRCA1 and BARD1 together form a heterodimer that exhibits E3 ubiquitin ligase activity in vitro but it remains controversial whether this enzymatic activity is essential for BRCA1-BARD1 in vivo function. To answer these questions, I took advantage of C. elegans, a small nematode that contains evolutionarily conserved BRCA1 and BARD1 orthologs (BRC-1 and BRD-1), to study their functions during spermatogenesis and oogenesis as well as to determine the requirement for E3 ligase activity of this complex in meiosis. By live cell imaging on worms expressing endogenous functional GFP fusions to BRC-1 and BRD-1, I found both proteins localize to DNA damage sites in early meiotic prophase and then concentrate onto a specific chromosome domain defined by crossover sites. Consistent with this localization pattern, I found BRC-1-BRD-1 plays important roles in DNA DSB repair and also influences crossover patterning in the germ line. During oogenesis, this complex stabilizes the RAD-51 filament in late pachytene and promotes the formation of extra crossovers under checkpoint activation conditions. Surprisingly, BRC-1-BRD-1 exhibits opposing functions during spermatogenesis, promoting DNA resection in early pachytene and inhibiting extra crossover formation when meiosis is perturbed. Using a combination of biochemistry and cell biology, I discovered that E3 ligase activity of BRC-1-BRD-1 is specifically required for the recruitment and concentration of the complex to DNA damage sites in the meiotic region of the gonad but not in the mitotic region where germline stem cells actively divide. The physiological substrates of BRC-1-BRD-1 in the germ line remain unknown, and future investigation will determine whether the differential functions observed for BRC-1- BRD-1 during oogenesis and spermatogenesis are results of the complex ubiquitylating different substrates in the female and male germ lines.