Identification of Rad51 regulation by BRCA2 using Caenorhabditis elegans BRCA2 and bimolecular fluorescence complementation analysis

Identification of Rad51 regulation by BRCA2 using Caenorhabditis elegans BRCA2 and bimolecular... BRCA2 is involved in double-stranded DNA break repair by binding and regulating Rad51-mediated homologous recombination. Insights as to how BRCA2 regulates Rad51-mediated DNA repair arose from in vitro biochemical studies on fragments of BRCA2. However, the large 400-kDa BRCA2 protein has hampered our ability to understand the entire process by which full-length BRCA2 regulates Rad51. Here, we show that CeBRC-2, which is only one tenth the size of mammalian BRCA2, complemented BRCA2-deficiency in Rad51 regulation. CeBRC-2 was able to bind to mammalian Rad51 (mRad51) and form distinct nuclear foci when they interacted. In our bimolecular fluorescence complementation analysis (BiFC), we show that the strength of the interaction between CeBRC-2 and mRad51 increased markedly after DNA damage. The BRC motif of CeBRC-2 was responsible for binding mRad51, but without the OB fold, the complex was unable to target damaged DNA. When CeBRC-2 was introduced into BRCA2-deficient cells, it restored Rad51 foci after DNA damage. Our study suggests a mode of action for BRCA2 with regard to DNA repair. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Biochemical and Biophysical Research Communications Elsevier

Identification of Rad51 regulation by BRCA2 using Caenorhabditis elegans BRCA2 and bimolecular fluorescence complementation analysis

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Abstract

BRCA2 is involved in double-stranded DNA break repair by binding and regulating Rad51-mediated homologous recombination. Insights as to how BRCA2 regulates Rad51-mediated DNA repair arose from in vitro biochemical studies on fragments of BRCA2. However, the large 400-kDa BRCA2 protein has hampered our ability to understand the entire process by which full-length BRCA2 regulates Rad51. Here, we show that CeBRC-2, which is only one tenth the size of mammalian BRCA2, complemented BRCA2-deficiency in Rad51 regulation. CeBRC-2 was able to bind to mammalian Rad51 (mRad51) and form distinct nuclear foci when they interacted. In our bimolecular fluorescence complementation analysis (BiFC), we show that the strength of the interaction between CeBRC-2 and mRad51 increased markedly after DNA damage. The BRC motif of CeBRC-2 was responsible for binding mRad51, but without the OB fold, the complex was unable to target damaged DNA. When CeBRC-2 was introduced into BRCA2-deficient cells, it restored Rad51 foci after DNA damage. Our study suggests a mode of action for BRCA2 with regard to DNA repair.

Journal

Biochemical and Biophysical Research CommunicationsElsevier

Published: Nov 3, 2007

References

  • Developmental studies of Brca1 and Brca2 knock-out mice
    Hakem, R.; de la Pompa, J.L.; Mak, T.W.
  • Sequence fingerprints in BRCA2 and RAD51: implications for DNA repair and cancer
    Lo, T.; Pellegrini, L.; Venkitaraman, A.R.; Blundell, T.L.
  • CeBRC-2 stimulates D-loop formation by RAD-51 and promotes DNA single-strand annealing
    Petalcorin, M.I.; Sandall, J.; Wigley, D.B.; Boulton, S.J.
  • Chromosome stability, DNA recombination and the BRCA2 tumour suppressor
    Venkitaraman, A.R.
  • From RPA to BRCA2: lessons from single-stranded DNA binding by the OB-fold
    Bochkarev, A.; Bochkareva, E.

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