Abstract
Mutations in BRCA2 increase susceptibility to breast, ovarian and prostate cancers. The product of human BRCA2, BRCA2 protein, has a key role in the repair of DNA double-strand breaks and interstrand cross-links by RAD51-mediated homologous recombination. Here, we present a biochemical and structural characterization of full-length (3,418 amino acid) BRCA2, alone and in complex with RAD51. We show that BRCA2 facilitates nucleation of RAD51 filaments at multiple sites on single-stranded DNA. Three-dimensional EM reconstructions revealed that BRCA2 exists as a dimer and that two oppositely oriented sets of RAD51 molecules bind the dimer. Single-stranded DNA binds along the long axis of BRCA2, such that only one set of RAD51 monomers can form a productive complex with DNA and establish filament formation. Our data define the molecular mechanism by which this tumor suppressor facilitates RAD51-mediated homologous-recombinational repair.
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Acknowledgements
We thank R. Carzaniga and L. Collinson at the London Research Institute Electron Microscope facility for advice and input and our colleagues for their comments. We thank Innova Biosciences for providing the InnovaCoat conjugation kit. This work was supported by the UK Medical Research Council and the Wellcome Trust (X.Z.) and by Cancer Research UK, the European Research Council, the Breast Cancer Campaign, Swiss Bridge and the Louis-Jeantet foundation (S.C.W.). L.M. was supported in part by an European Molecular Biology Organization Fellowship. J.S. was supported in part by a Marie Curie Intra-European Fellowship (MC-IEF-625826).
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S.C.W. and X.Z. conceived the original ideas for this study. All authors contributed to experimental designs. T.S., J.S., E.H.K. and M.J.M. performed the experiments. L.M. and T.P. contributed to initial characterizations of the BRCA2 protein. T.S., J.S., E.H.K., M.J.M., S.C.W. and X.Z. analyzed the data. X.Z. and S.C.W. wrote the main text, and all authors contributed to the writing of the paper.
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Supplementary Figure 1 BRCA2 and BRCA2–RAD51 purification and binding to DNA substrates.
(a) SDS-PAGE showing the purified proteins used for the electron microscopy studies. (b) BRCA2 stimulates RAD51-ssDNA nucleoprotein filament formation, as determined by gel-shift assays. (c) Binding of BRCA2 to gapped DNA, as determined by gel-shift assays.
Supplementary Figure 2 Electron micrographs of BRCA2 and BRCA2–RAD51 complex.
(a) Micrograph and representative selected individual particles of BRCA2. (b) Micrograph and representative selected individual particles of BRCA2-RAD51. Scale bar is 100 nm.
Supplementary Figure 3 Image processing of BRCA2 reconstruction and antibody labeling.
(a) Initial eigen-images show a clear 2-fold component in the particles. (b) Non-symmetrized 3D reconstruction in orthogonal reprojections (top) and slices though the 3D (bottom) showing 2-fold component (c) Representative class averages (CA) and reprojections from the final 3D reconstruction. (d) Locations of antibody against Flag tag (blue) and BRC repeats (magenta) through triangulation for top and side views.
Supplementary Figure 4 Resolution of EM reconstructions and tilt validation.
(a) Resolution as determined by Fourier shell correlation coefficient using 0.5, ½-bit and 0.143 criteria for binary masking (bm), soft spherical masking (csm) or no masking (nm) applied to the BRCA2 and BRCA2-RAD51 reconstructions. On the right are the corresponding Euler angle distributions of classums that gave rise to the final 3D reconstructions. (b) Particles every 10º from a 0-40º tilt-series with 5º steps and matching projections of BRCA2 (left) and BRCA2-RAD51 (right), and (c) tables of nominal versus measured tilts for BRCA2 (left) and BRCA2-RAD51 (right).
Supplementary Figure 5 Image processing of BRCA2–RAD51 reconstruction.
(a) Initial eigenimages show a clear 2-fold component in the particles. (b) Non-symmetrized 3D reconstruction in orthogonal reprojections (top) and slices though the 3D (bottom) showing 2-fold component (c) Representative class averages (CA) and reprojections from the 3D reconstruction.
Supplementary Figure 6 RecA and RAD51 filament orientations.
(a) In addition to the conserved ATPase core domain, RecA and RAD51 have distinct additional domains positioned on opposite sides of the ATPase core relative to ssDNA. (b) Schematic diagram of how RecA (yellow and red) and RAD51 (yellow and blue) subunits may be added to the growing filament. The ATPase domain is represented in yellow. The respective loops that only become ordered and stabilized in the filament are shown as broken (disordered) and solid lines (ordered). (c) RecA-ssDNA filament as observed in the crystal structure and RAD51 filament modelled onto the RecA filament. The unique domains are boxed.
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Shahid, T., Soroka, J., Kong, E. et al. Structure and mechanism of action of the BRCA2 breast cancer tumor suppressor. Nat Struct Mol Biol 21, 962–968 (2014). https://doi.org/10.1038/nsmb.2899
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DOI: https://doi.org/10.1038/nsmb.2899
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