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Purified human BRCA2 stimulates RAD51-mediated recombination

Abstract

Mutation of the breast cancer susceptibility gene, BRCA2, leads to breast and ovarian cancers. Mechanistic insight into the functions of human BRCA2 has been limited by the difficulty of isolating this large protein (3,418 amino acids). Here we report the purification of full-length BRCA2 and show that it both binds RAD51 and potentiates recombinational DNA repair by promoting assembly of RAD51 onto single-stranded DNA (ssDNA). BRCA2 acts by targeting RAD51 to ssDNA over double-stranded DNA, enabling RAD51 to displace replication protein-A (RPA) from ssDNA and stabilizing RAD51–ssDNA filaments by blocking ATP hydrolysis. BRCA2 does not anneal ssDNA complexed with RPA, implying it does not directly function in repair processes that involve ssDNA annealing. Our findings show that BRCA2 is a key mediator of homologous recombination, and they provide a molecular basis for understanding how this DNA repair process is disrupted by BRCA2 mutations, which lead to chromosomal instability and cancer.

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Figure 1: Protein interactions with purified full-length human BRCA2.
Figure 2: BRCA2 displays a strong preference for binding tailed and ssDNA substrates over dsDNA.
Figure 3: BRCA2 stimulates DNA strand exchange promoted by RAD51.
Figure 4: BRCA2 stimulates RAD51-mediated DNA strand exchange by promoting stable RAD51–ssDNA filament formation, overcoming inhibition by RPA.
Figure 5: BRCA2 does not anneal ssDNA complexed with RPA.

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Acknowledgements

We thank D. Shin for BRCA2 cDNA clones; M. Zdzienicka for VC8 cells; A. Mazin for RAD52; A. Nimonkar for DMC1; W. Heyer and Kowalczykowski laboratory for comments. Supported by grants from NIH (NIH GM 62653) and DOD-Breast Cancer Research Program (BC085223) to S.C.K., American Cancer Society Postdoctoral Fellowship to R.B.J. (PF-05-225-01-GMC), and Postdoctoral Fellowship from Ministerio de Educación y Ciencia (Spain) to A.C.; R.B.J. acknowledges financial support from C. Hornung, who funded this work through the American Cancer Society and who passed away last year.

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R.B.J. and S.C.K. conceived the general ideas for this study. R.B.J., A.C. and S.C.K. planned experiments and interpreted data; R.B.J. and A.C. performed the experiments. R.B.J. and S.C.K. wrote the manuscript and all authors provided editorial input.

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Correspondence to Stephen C. Kowalczykowski.

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Jensen, R., Carreira, A. & Kowalczykowski, S. Purified human BRCA2 stimulates RAD51-mediated recombination. Nature 467, 678–683 (2010). https://doi.org/10.1038/nature09399

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