RAD-51-Dependent and -Independent Roles of a Caenorhabditis elegans BRCA2-Related Protein during DNA Double-Strand Break Repair

Abstract

The BRCA2 tumor suppressor is implicated in DNA double-strand break (DSB) repair by homologous recombination (HR), where it regulates the RAD51 recombinase. We describe a BRCA2-related protein of Caenorhabditis elegans (CeBRC-2) that interacts directly with RAD-51 via a single BRC motif and that binds preferentially to single-stranded DNA through an oligonucleotide-oligosaccharide binding fold. Cebrc-2 mutants fail to repair meiotic or radiation-induced DSBs by HR due to inefficient RAD-51 nuclear localization and a failure to target RAD-51 to sites of DSBs. Genetic and cytological comparisons of Cebrc-2 and rad-51 mutants revealed fundamental phenotypic differences that suggest a role for Cebrc-2 in promoting the use of an alternative repair pathway in the absence of rad-51 and independent of nonhomologous end joining (NHEJ). Unlike rad-51 mutants, Cebrc-2 mutants also accumulate RPA-1 at DSBs, and abnormal chromosome aggregates that arise during the meiotic prophase can be rescued by blocking the NHEJ pathway. CeBRC-2 also forms foci in response to DNA damage and can do so independently of rad-51. Thus, CeBRC-2 not only regulates RAD-51 during HR but can also function independently of rad-51 in DSB repair processes. 1 The faithful segregation of homologous chromosomes during meiosis is dependent on the formation of physical connections (chiasmata) that form following the successful reciprocal exchange of DNA molecules resulting from crossover recombination. In concert with sister chromatid cohesion, chiasmata facilitate the proper orientation of homologous chromosomes and their subsequent segregation to opposite poles of the meiotic spindle (53). In most eukaryotes, meiotic recombination is initiated by the action of Spo11, a topoisomerase II-like enzyme that catalyzes the formation of meiosis-specific DNA double-strand breaks (DSBs) (15,18). Once formed, a DSB is processed to generate a resected 3Ј single-stranded DNA (ssDNA) tail that is rapidly bound by replication protein A (RPA) before being displaced by RAD51, the eukaryotic homolog of the bacterial DNA strand exchange protein RecA. Rad51 catalyzes DNA strand exchange between homologous sequences, thus promoting the physical exchange of DNA molecules required for successful crossover recombination.

BRCA2: a universal recombinase regulator

“…Perhaps surprisingly, orthologues of the BRCA2 gene have been found in a variety of evolutionary diverse taxa (Figure 4a), such as the smut fungus Ustilago maydis (UmBRH2) (Kojic et al, 2002), the nematode Caenorhabditis elegans (CeBRC-2) (Martin et al, 2005), the plant Arabidopsis thaliana (AtBRCA2) (Siaud et al, 2004), as well as vertebrates such as chicken (cBRCA2) (Takata et al, 2002) (Figure 4a). Human BRCA2 is a very large polypeptide of 3418 amino acids and it contains a number of recognizable motifs, some of which can be found in all BRCA2 homologues.…”

“…In the nematode C. elegans, a null mutation in BRC-2 results in defects in meiotic recombination (Martin et al, 2005). Similarly, evidence for a role in meiosis came from plants.…”

Interaction between Arabidopsis Brca2 and Its Partners Rad51, Dmc1, and Dss1

“…However, the ability of Brca2 to associate with Rad51 is conserved in Ustilago, Arabidopsis, and C. elegans. In Arabidopsis, two proteins of 1,151/1,155 amino acids, with four BRC repeats each, can be defined: AtBrca2(IV) and AtBrca2(V) that are 94.5% identical (Siaud et al, 2004;Kojic et al, 2005;Martin et al, 2005). Studies considering the role of Brca2 in organisms other than mammals offered interesting clues about its function in vivo, especially since Brca2-depleted Ustilago, Arabidopsis, or Caenorhabditis were viable.…”

Meiotic double strand breaks repair in sexually reproducing eukaryotes: We are not all equal

“…However in both organisms the brc-2 gene (orthologous to the breast cancer susceptibility gene BRCA2 and absent in fungi) is essential for RecA-homologues loading at DSBs sites [24,16]. In nematodes, brc-2 and rad-51 mutants exhibit a similar phenotype resulting in extensive chromatin decondensation/fusion at diakinesis [16,24]. In absence of BRC-2, DNA ends appear over-resected [24].…”

“…In nematodes, brc-2 and rad-51 mutants exhibit a similar phenotype resulting in extensive chromatin decondensation/fusion at diakinesis [16,24]. In absence of BRC-2, DNA ends appear over-resected [24]. Furthermore, BRC-2 stimulates RAD-51-mediated D-loop formation and reduces the rate of ATP hydrolysis catalyzed by RAD-51 suggesting that BRC-2 can also prevent RAD-51 nucleoprotein filament depolymerization [25,26].…”