CeBRC-2 stimulates D-loop formation by RAD-51 and promotes DNA single-strand annealing |
| |
Authors: | Petalcorin Mark I R Sandall Jane Wigley Dale B Boulton Simon J |
| |
Affiliation: | Molecular Enzymology Laboratory, Cancer Research UK, The London Research Institute, Clare Hall Laboratories, South Mimms, EN6 3LD, UK. |
| |
Abstract: | 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. |
| |
Keywords: | DSB, DNA double-strand break NHEJ, non-homologous end joining SSA, single-strand annealing HR, homologous recombination ssDNA, single-stranded DNA dsDNA, double-stranded DNA OB, oligonucleotide/oligosaccharide-binding WT, wild-type |
本文献已被 ScienceDirect PubMed 等数据库收录! |
|