CeBRC-2 Stimulates D-loop Formation by RAD-51 and Promotes DNA Single-strand Annealing

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. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Molecular Biology Elsevier

CeBRC-2 Stimulates D-loop Formation by RAD-51 and Promotes DNA Single-strand Annealing

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Publisher
Elsevier
Copyright
Copyright © 2006 Elsevier Ltd
ISSN
0022-2836
D.O.I.
10.1016/j.jmb.2006.06.020
Publisher site
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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.

Journal

Journal of Molecular BiologyElsevier

Published: Aug 11, 2006

References

  • Double-strand break repair in human cells
    West, S.C.; Chappell, C.; Hanakahi, L.A.; Masson, J.Y.; McIlwraith, M.J.; Van Dyck, E.
  • Human Rad52 protein promotes single-strand DNA annealing followed by branch migration
    Reddy, G.; Golub, E.I.; Radding, C.M.
  • Rad52 forms ring structures and co-operates with RPA in single-strand DNA annealing
    Shinohara, A.; Shinohara, M.; Ohta, T.; Matsuda, S.; Ogawa, T.
  • DSS1 is required for RAD51 focus formation and genomic stability in mammalian cells
    Gudmundsdottir, K.; Lord, C.J.; Witt, E.; Tutt, A.N.; Ashworth, A.
  • Brca2 is involved in meiosis in Arabidopsis thaliana as suggested by its interaction with Dmc1
    Siaud, N.; Dray, E.; Gy, I.; Gerard, E.; Takvorian, N.; Doutriaux, M.P.
  • New complexities for BRCA1 and BRCA2
    Kerr, P.; Ashworth, A.
  • Roles for Caenorhabditis elegans rad-51 in meiosis and in resistance to ionizing radiation during development
    Rinaldo, C.; Bazzicalupo, P.; Ederle, S.; Hilliard, M.; La Volpe, A.
  • Mutation in Brca2 stimulates error-prone homology-directed repair of DNA double-strand breaks occurring between repeated sequences
    Tutt, A.; Bertwistle, D.; Valentine, J.; Gabriel, A.; Swift, S.; Ross, G.
  • Reconstitution of the strand invasion step of double-strand break repair using human Rad51 Rad52 and RPA proteins
    McIlwraith, M.J.; Van Dyck, E.; Masson, J.Y.; Stasiak, A.Z.; Stasiak, A.; West, S.C.

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