CeBRC-2 stimulates D-loop formation by RAD-51 and promotes DNA single-strand annealing.
The BRCA2 tumour suppressor regulates the RAD-51 recombinase during double-strand break (DSB) repair by homologous recombination (HR) but how BRCA2 executes its functions is not well understood. We previously described a functional homologue of BRCA2 in Caenorhabditis elegans (CeBRC-2) that binds preferentially to single-stranded DNA via an OB-fold domain and associates directly with RAD-51 via a single BRC domain. Consistent with a direct role in HR, Cebrc-2 mutants are defective for repair of meiotic and radiation-induced DSBs due to an inability to regulate RAD-51. Here, we explore the function of CeBRC-2 in HR processes using purified proteins. We show that CeBRC-2 stimulates RAD-51-mediated D-loop formation and reduces the rate of ATP hydrolysis catalysed by RAD-51. These functions of CeBRC-2 are dependent upon direct association with RAD-51 via its BRC motif and on its DNA-binding activity, as point mutations in the BRC domain that abolish RAD-51 binding or the BRC domain of CeBRC-2 alone, lacking the DNA-binding domain, fail to stimulate RAD-51-mediated D-loop formation and do not reduce the rate of ATP hydrolysis by RAD-51. Phenotypic comparison of Cebrc-2 and rad-51 mutants also revealed a role for CeBRC-2 in an error-prone DSB repair pathway independent of rad-51 and non-homologous end joining, raising the possibility that CeBRC-2 may have replaced the role of vertebrate Rad52 in DNA single-strand annealing (SSA), which is missing from C. elegans. Indeed, we show here that CeBRC-2 mediates SSA of RPA-oligonucleotide complexes similar to Rad52. These results reveal RAD-51-dependent and -independent functions of CeBRC-2 that provide an explanation for the difference in DNA repair defects observed in Cebrc-2 and rad-51 mutants, and define mechanistic roles for CeBRC-2 in HR and in the SSA pathway for DSB repair.
Petalcorin
Mark I R
MI
Molecular Enzymology Laboratory, Cancer Research UK, The London Research Institute, Clare Hall Laboratories, South Mimms, EN6 3LD, UK.
Sandall
Jane
J
Wigley
Dale B
DB
Boulton
Simon J
SJ
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D017173:Caenorhabditis elegans / Q000502:physiology; D029742:Caenorhabditis elegans Proteins / Q000235:genetics / Q000378:metabolism*; D004249:DNA Damage; D004260:DNA Repair / Q000502:physiology*; D004277:DNA, Single-Stranded / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D006868:Hydrolysis; D008969:Molecular Sequence Data; D051135:Rad51 Recombinase / Q000235:genetics / Q000378:metabolism*; D011995:Recombination, Genetic
C500610:BRC-2 protein, C elegans; D029742:Caenorhabditis elegans Proteins; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D000255:Adenosine Triphosphate; D051135:Rad51 Recombinase; C495148:rad-51 protein, C elegans