Differential requirement for BRCA1-BARD1 E3 ubiquitin ligase activity in DNA damage repair and meiosis in the Caenorhabditis elegans germ line

Abstract

The tumor suppressor BRCA1-BARD1 complex functions in many cellular processes; of critical importance to its tumor suppressor function is its role in genome integrity. Although RING E3 ubiquitin ligase activity is the only known enzymatic activity of the complex, the in vivo requirement for BRCA1-BARD1 E3 ubiquitin ligase activity has been controversial. Here we probe the role of BRCA1-BARD1 E3 ubiquitin ligase activity in vivo using C. elegans. Genetic, cell biological, and biochemical analyses of mutants defective for E3 ligase activity reveal both E3 ligase-dependent and independent functions of the complex in the context of DNA damage repair and meiosis. We show that E3 ligase activity is essential for BRCA1-BARD1 to concentrate at both DNA damage and recombination sites in meiotic germ cells, but not at DNA damage sites in proliferating germ cells. While BRCA1 alone is capable of monoubiquitylation, BARD1 is required with BRCA1 to promote polyubiquitylation. We find that the requirement for E3 ligase activity and BARD1 in DNA damage signaling and repair can be partially alleviated by driving the nuclear accumulation and self-association of BRCA1. Our data suggest that in addition to E3 ligase activity, BRC-1 serves a structural role for DNA damage signaling and repair while BRD-1 plays an accessory role to enhance BRC-1 function. Author SummaryBRCA1-BARD1 is a E3 ubiquitin ligase, which modifies proteins by the addition of the small protein ubiquitin. While mutations that disrupt E3 ligase activity and stability of the BRCA1-BARD1 complex lead to a predisposition for breast and ovarian cancer, the specific requirement for E3 ligase activity in tumor suppression is not known. Here we probe the function of E3 ligase activity and BARD1 in the maintenance of genome integrity by engineering point mutations that disrupt E3 ligase activity in C. elegans BRCA1 as well as a null mutation in BARD1. We find that while E3 ligase activity is important for genome integrity, the complex plays additional roles besides ubiquitylating proteins. Further, our data suggest that BRCA1 is the key functional unit of the complex while BARD1 is an accessory partner that enhances BRCA1s function. These findings may help explain why there is a higher prevalence of cancer-causing mutations in BRCA1 compared to BARD1.