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Structure and mechanism of action of the BRCA2 breast cancer tumor suppressor

Nature Structural & Molecular Biology volume 21, pages 962–968 (2014)Cite this article

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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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Figure 1: 3D reconstruction of BRCA2 and identification of domains.
Figure 2: 3D reconstruction of BRCA2–RAD51 complex.
Figure 3: RAD51 binding to BRCA2.
Figure 4: ssDNA binding of BRCA2 and BRCA2–RAD51 complex.
Figure 5: Nucleation of RAD51 filaments by BRCA2.
Figure 6: A proposed mode of action for BRCA2 in RAD51 filament nucleation.

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References

  1. Stratton, M.R. & Rahman, N. The emerging landscape of breast cancer susceptibility. Nat. Genet. 40, 17–22 (2008).

    Article  CAS  PubMed  Google Scholar 

  2. Wooster, R. et al. Identification of the breast cancer susceptibility gene BRCA2. Nature 378, 789–792 (1995).

    Article  CAS  PubMed  Google Scholar 

  3. King, M.C., Marks, J.H., Mandell, J.B. & Grp, N.Y.B.C.S. Breast and ovarian cancer risks due to inherited mutations in BRCA1 and BRCA2. Science 302, 643–646 (2003).

    Article  CAS  PubMed  Google Scholar 

  4. Lindahl, T. Instability and decay of the primary structure of DNA. Nature 362, 709–715 (1993).

    Article  CAS  PubMed  Google Scholar 

  5. Jackson, S.P. & Bartek, J. The DNA-damage response in human biology and disease. Nature 461, 1071–1078 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Venkitaraman, A.R. Cancer susceptibility and the functions of BRCA1 and BRCA2. Cell 108, 171–182 (2002).

    Article  CAS  PubMed  Google Scholar 

  7. Roy, R., Chun, J. & Powell, S.N. BRCA1 and BRCA2: different roles in a common pathway of genome protection. Nat. Rev. Cancer 12, 68–78 (2012).

    Article  CAS  Google Scholar 

  8. Moynahan, M.E., Pierce, A.J. & Jasin, M. BRCA2 is required for homology-directed repair of chromosomal breaks. Mol. Cell 7, 263–272 (2001).

    Article  CAS  PubMed  Google Scholar 

  9. Lomonosov, M., Anand, S., Sangrithi, M., Davies, R. & Venkitaraman, A.R. Stabilization of stalled DNA replication forks by the BRCA2 breast cancer susceptibility protein. Genes Dev. 17, 3017–3022 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Schlacher, K. et al. Double-strand break repair-independent role for BRCA2 in blocking stalled replication fork degradation by MRE11. Cell 145, 529–542 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Yuan, S.-S.F. et al. BRCA2 is required for ionizing radiation-induced assembly of RAD51 complex in vivo. Cancer Res. 59, 3547–3551 (1999).

    CAS  PubMed  Google Scholar 

  12. Tarsounas, M., Davies, D. & West, S.C. BRCA2-dependent and independent formation of RAD51 nuclear foci. Oncogene 22, 1115–1123 (2003).

    Article  CAS  PubMed  Google Scholar 

  13. Bork, P., Blomberg, N. & Nilges, M. Internal repeats in the BRCA2 protein sequence. Nat. Genet. 13, 22–23 (1996).

    Article  CAS  PubMed  Google Scholar 

  14. Bignell, G., Micklem, G., Stratton, M.R., Ashworth, A. & Wooster, R. The BRC repeats are conserved in mammalian BRCA2 proteins. Hum. Mol. Genet. 6, 53–58 (1997).

    Article  CAS  PubMed  Google Scholar 

  15. Davies, A.A. et al. Role of BRCA2 in control of the RAD51 recombination and DNA repair protein. Mol. Cell 7, 273–282 (2001).

    Article  CAS  PubMed  Google Scholar 

  16. Esashi, F., Galkin, V.E., Yu, X., Egelman, E.H. & West, S.C. Stabilization of RAD51 nucleoprotein filaments by the C-terminal region of BRCA2. Nat. Struct. Mol. Biol. 14, 468–474 (2007).

    Article  CAS  PubMed  Google Scholar 

  17. Carreira, A. et al. The BRC repeats of BRCA2 modulate the DNA-binding specificity of RAD51. Cell 136, 1032–1043 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Pellegrini, L. et al. Insights into DNA recombination from the structure of a RAD51–BRCA2 complex. Nature 420, 287–293 (2002).

    Article  CAS  PubMed  Google Scholar 

  19. Tal, A., Arbel-Goren, R. & Stavans, J. Cancer-associated mutations in BRC domains of BRCA2 affect homologous recombination induced by RAD51. J. Mol. Biol. 393, 1007–1012 (2009).

    Article  CAS  PubMed  Google Scholar 

  20. Thorslund, T. et al. The breast cancer tumor suppressor BRCA2 promotes the specific targeting of RAD51 to single-stranded DNA. Nat. Struct. Mol. Biol. 17, 1263–1265 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Jensen, R.B., Carreira, A. & Kowalczykowski, S.C. Purified human BRCA2 stimulates RAD51-mediated recombination. Nature 467, 678–683 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Liu, J., Doty, T., Gibson, B. & Heyer, W.D. Human BRCA2 protein promotes RAD51 filament formation on RPA-covered single-stranded DNA. Nat. Struct. Mol. Biol. 17, 1260–1262 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Lee, S.A., Roques, C., Magwood, A.C., Masson, J.Y. & Baker, M.D. Recovery of deficient homologous recombination in BRCA2-depleted mouse cells by wild-type RAD51 expression. DNA Repair (Amst.) 8, 170–181 (2009).

    Article  CAS  Google Scholar 

  24. Buisson, R. et al. Cooperation of breast cancer proteins PALB2 and piccolo BRCA2 in stimulating homologous recombination. Nat. Struct. Mol. Biol. 17, 1247–1254 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Dray, E. et al. Enhancement of RAD51 recombinase activity by the tumor suppressor PALB2. Nat. Struct. Mol. Biol. 17, 1255–1259 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Rahman, N. et al. PALB2, which encodes a BRCA2-interacting protein, is a breast cancer susceptibility gene. Nat. Genet. 39, 165–167 (2007).

    Article  CAS  PubMed  Google Scholar 

  27. Sy, S.M.H., Huen, M.S.Y., Zhu, Y.Y. & Chen, J.J. PALB2 regulates recombinational repair through chromatin association and oligomerization. J. Biol. Chem. 284, 18302–18310 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Thorslund, T., Esashi, F. & West, S.C. Interactions between human BRCA2 protein and the meiosis-specific recombinase DMC1. EMBO J. 26, 2915–2922 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Yang, H. et al. BRCA2 function in DNA binding and recombination from a BRCA2-DSS1-ssDNA structure. Science 297, 1837–1848 (2002).

    Article  CAS  PubMed  Google Scholar 

  30. van Heel, M. et al. Single-particle electron cryo-microscopy: towards atomic resolution. Q. Rev. Biophys. 33, 307–369 (2000).

    Article  CAS  PubMed  Google Scholar 

  31. van Heel, M., Harauz, G., Orlova, E.V., Schmidt, R. & Schatz, M. A new generation of the IMAGIC image processing system. J. Struct. Biol. 116, 17–24 (1996).

    Article  CAS  PubMed  Google Scholar 

  32. Rosenthal, P.B. & Henderson, R. Optimal determination of particle orientation, absolute hand, and contrast loss in single-particle electron cryomicroscopy. J. Mol. Biol. 333, 721–745 (2003).

    Article  CAS  PubMed  Google Scholar 

  33. Sauerwald, A. et al. Structure of active dimeric human telomerase. Nat. Struct. Mol. Biol. 20, 454–460 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Wong, A.K.C., Pero, R., Ormonde, P.A., Tavtigian, S.V. & Bartel, P.L. RAD51 interacts with the evolutionarily conserved BRC motifs in the human breast cancer susceptibility gene BRCA2. J. Biol. Chem. 272, 31941–31944 (1997).

    Article  CAS  PubMed  Google Scholar 

  35. Goddard, T.D., Huang, C.C. & Ferrin, T.E. Visualizing density maps with UCSF Chimera. J. Struct. Biol. 157, 281–287 (2007).

    Article  CAS  PubMed  Google Scholar 

  36. Benson, F.E., Stasiak, A. & West, S.C. Purification and characterisation of the human RAD51 protein, an analogue of E. coli RecA. EMBO J. 13, 5764–5771 (1994).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Baumann, P., Benson, F.E. & West, S.C. Human RAD51 protein promotes ATP-dependent homologous pairing and strand transfer reactions in vitro. Cell 87, 757–766 (1996).

    Article  CAS  PubMed  Google Scholar 

  38. Shivji, M.K.K. et al. The BRC repeats of human BRCA2 differentially regulate RAD51 binding on single- versus double-stranded DNA to stimulate strand exchange. Proc. Natl. Acad. Sci. USA 106, 13254–13259 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Ho, Y. et al. Systematic identification of protein complexes in Saccharomyces cerevisiae by mass spectrometry. Nature 415, 180–183 (2002).

    Article  CAS  PubMed  Google Scholar 

  40. Sung, P. & Robberson, D.L. DNA strand exchange mediated by a RAD51-ssDNA nucleoprotein filament with polarity opposite to that of RecA. Cell 82, 453–461 (1995).

    Article  CAS  PubMed  Google Scholar 

  41. Baumann, P. & West, S.C. The human RAD51 protein: polarity of strand transfer and stimulation by hRP-A. EMBO J. 16, 5198–5206 (1997).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Hilario, J., Amitani, I., Baskin, R.J. & Kowalczykowski, S.C. Direct imaging of human RAD51 nucleoprotein dynamics on individual DNA molecules. Proc. Natl. Acad. Sci. USA 106, 361–368 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. van der Heijden, T. et al. Real-time assembly and disassembly of human RAD51 filaments on individual DNA molecules. Nucleic Acids Res. 35, 5646–5657 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Zhou, Q. et al. Dss1 interaction with Brh2 as a regulatory mechanism for recombinational repair. Mol. Cell. Biol. 27, 2512–2526 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Jeyasekharan, A.D. et al. DNA damage regulates the mobility of BRCA2 within the nucleoplasm of living cells. Proc. Natl. Acad. Sci. USA 107, 21937–21942 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Bakkenist, C.J. & Kastan, M.B. DNA damage activates ATM through intermolecular autophosphorylation and dimer dissociation. Nature 421, 499–506 (2003).

    Article  CAS  PubMed  Google Scholar 

  47. Saeki, H. et al. Suppression of the DNA repair defects of BRCA2-deficient cells with heterologous protein fusions. Proc. Natl. Acad. Sci. USA 103, 8768–8773 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Bochkarev, A. & Bochkareva, E. From RPA to BRCA2: lessons from single-stranded DNA binding by the OB-fold. Curr. Opin. Struct. Biol. 14, 36–42 (2004).

    Article  CAS  PubMed  Google Scholar 

  49. Fan, J. & Pavletich, N.P. Structure and conformational change of a replication protein A heterotrimer bound to ssDNA. Genes Dev. 26, 2337–2347 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Bianchi, M., Das Gupta, C. & Radding, C.M. Synapsis and the formation of paranemic joints by E. coli RecA protein. Cell 34, 931–939 (1983).

    Article  CAS  PubMed  Google Scholar 

  51. Bortner, C. & Griffith, J. Three-stranded paranemic joints: architecture, topological constraints and movement. J. Mol. Biol. 215, 623–634 (1990).

    Article  CAS  PubMed  Google Scholar 

  52. McIlwraith, M.J. et al. Reconstitution of the strand invasion step of double-strand break repair using human RAD51, RAD52 and RPA proteins. J. Mol. Biol. 304, 151–164 (2000).

    Article  CAS  PubMed  Google Scholar 

  53. Namsaraev, E.A. & Berg, P. Rad51 uses one mechanism to drive DNA strand exchange in both directions. J. Biol. Chem. 275, 3970–3976 (2000).

    Article  CAS  PubMed  Google Scholar 

  54. Yang, H., Li, Q., Holloman, W.K. & Pavletich, N.P. The BRCA2 homologue Brh2 nucleates RAD51 filament formation at a dsDNA-ssDNA junction. Nature 433, 653–657 (2005).

    Article  CAS  PubMed  Google Scholar 

  55. West, S.C., Cassuto, E. & Howard-Flanders, P. Heteroduplex formation by RecA protein: polarity of strand exchange. Proc. Natl. Acad. Sci. USA 78, 6149–6153 (1981).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Kahn, R., Cunningham, R.P., DasGupta, C. & Radding, C.M. Polarity of heteroduplex formation promoted by Escherichia coli RecA protein. Proc. Natl. Acad. Sci. USA 78, 4786–4790 (1981).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Cox, M.M. & Lehman, I.R. Directionality and polarity in RecA protein-promoted branch migration. Proc. Natl. Acad. Sci. USA 78, 6018–6022 (1981).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Baumann, P., Benson, F.E., Hajibagheri, N. & West, S.C. Purification of human RAD51 protein by selective spermidine precipitation. Mutat. Res. DNA Repair 384, 65–72 (1997).

    Article  CAS  PubMed  Google Scholar 

  59. Rosenthal, P.B. & Henderson, R. Optimal determination of particle orientation, absolute hand, and contrast loss in single-particle electron cryomicroscopy. J. Mol. Biol. 333, 721–745 (2003).

    Article  CAS  PubMed  Google Scholar 

  60. Henderson, R. et al. Tilt-pair analysis of images from a range of different specimens in single-particle electron cryomicroscopy. J. Mol. Biol. 413, 1028–1046 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  61. Sauerwald, A. et al. Structure of active dimeric human telomerase. Nat. Struct. Mol. Biol. 20, 454–460 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  62. Wall, J.S., Hainfeld, J.F. & Simon, M.N. Scanning transmission electron microscopy of nuclear structures. Methods Cell Biol. 53, 139–164 (1998).

    Article  CAS  PubMed  Google Scholar 

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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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Author notes

  1. Laurent Malivert

    Present address: Present address: Synthace Ltd., London Bioscience Innovation Centre, London, UK.,

  2. Taha Shahid, Joanna Soroka and Eric H Kong: These authors contributed equally to this work.

Authors and Affiliations

  1. Centre for Structural Biology, Imperial College, London, UK

    Taha Shahid, Eric H Kong, Tillmann Pape & Xiaodong Zhang

  2. London Research Institute, Clare Hall Laboratories, South Mimms, UK

    Joanna Soroka, Laurent Malivert, Michael J McIlwraith & Stephen C West

  3. Department of Medicine, Imperial College, London, UK

    Xiaodong Zhang

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Contributions

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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Correspondence to Stephen C West or Xiaodong Zhang.

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Integrated supplementary information

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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