Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

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.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

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.

References

  1. Lengauer, C., Kinzler, K. W. & Vogelstein, B. Genetic instabilities in human cancers. Nature 396, 643–649 (1998)

    Article  ADS  CAS  Google Scholar 

  2. Yu, V. P. et al. Gross chromosomal rearrangements and genetic exchange between nonhomologous chromosomes following BRCA2 inactivation. Genes Dev. 14, 1400–1406 (2000)

    CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  ADS  CAS  Google Scholar 

  4. Phelan, C. M. et al. Mutation analysis of the BRCA2 gene in 49 site-specific breast cancer families. Nature Genet. 13, 120–122 (1996)

    Article  CAS  Google Scholar 

  5. 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  Google Scholar 

  6. 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  Google Scholar 

  7. Esashi, F. et al. CDK-dependent phosphorylation of BRCA2 as a regulatory mechanism for recombinational repair. Nature 434, 598–604 (2005)

    Article  ADS  CAS  Google Scholar 

  8. Bianco, P. R., Tracy, R. B. & Kowalczykowski, S. C. DNA strand exchange proteins: a biochemical and physical comparison. Front. Biosci. 3, D570–D603 (1998)

    Article  CAS  Google Scholar 

  9. 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  Google Scholar 

  10. 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  Google Scholar 

  11. Chen, C. F., Chen, P. L., Zhong, Q., Sharp, Z. D. & Lee, W. H. Expression of BRC repeats in breast cancer cells disrupts the BRCA2-Rad51 complex and leads to radiation hypersensitivity and loss of G2/M checkpoint control. J. Biol. Chem. 274, 32931–32935 (1999)

    Article  CAS  Google Scholar 

  12. 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  Google Scholar 

  13. Yuan, S. S. 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 

  14. Godthelp, B. C., Artwert, F., Joenje, H. & Zdzienicka, M. Z. Impaired DNA damage-induced nuclear Rad51 foci formation uniquely characterizes Fanconi anemia group D1. Oncogene 21, 5002–5005 (2002)

    Article  CAS  Google Scholar 

  15. San Filippo, J. et al. Recombination mediator and Rad51 targeting activities of a human BRCA2 polypeptide. J. Biol. Chem. 281, 11649–11657 (2006)

    Article  CAS  Google Scholar 

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

    Article  ADS  CAS  Google Scholar 

  17. Yang, H., Li, Q., Fan, J., 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  ADS  CAS  Google Scholar 

  18. 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  Google Scholar 

  19. 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. Nature Struct. Mol. Biol. 14, 468–474 (2007)

    Article  CAS  Google Scholar 

  20. 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  ADS  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  22. Carreira, A. & Kowalczykowski, S. C. BRCA2: shining light on the regulation of DNA-binding selectivity by RAD51. Cell Cycle 8, 3445–3447 (2009)

    Article  CAS  Google Scholar 

  23. Petalcorin, M. I., Sandall, J., Wigley, D. B. & Boulton, S. J. CeBRC-2 stimulates D-loop formation by RAD-51 and promotes DNA single-strand annealing. J. Mol. Biol. 361, 231–242 (2006)

    Article  CAS  Google Scholar 

  24. Marmorstein, L. Y., Ouchi, T. & Aaronson, S. A. The BRCA2 gene product functionally interacts with p53 and RAD51. Proc. Natl Acad. Sci. USA 95, 13869–13874 (1998)

    Article  ADS  CAS  Google Scholar 

  25. Sharan, S. K. et al. Embryonic lethality and radiation hypersensitivity mediated by Rad51 in mice lacking Brca2 . Nature 386, 804–810 (1997)

    Article  ADS  CAS  Google Scholar 

  26. Chen, P. L. et al. The BRC repeats in BRCA2 are critical for RAD51 binding and resistance to methyl methanesulfonate treatment. Proc. Natl Acad. Sci. USA 95, 5287–5292 (1998)

    Article  ADS  CAS  Google Scholar 

  27. 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  Google Scholar 

  28. Shinohara, A. et al. Cloning of human, mouse and fission yeast recombination genes homologous to RAD51 and recA . Nature Genet. 4, 239–243 (1993)

    Article  CAS  Google Scholar 

  29. Yoshimura, Y., Morita, T., Yamamoto, A. & Matsushiro, A. Cloning and sequence of the human RecA-like gene cDNA. Nucleic Acids Res. 21, 1665 (1993)

    Article  CAS  Google Scholar 

  30. Wong, J. M., Ionescu, D. & Ingles, C. J. Interaction between BRCA2 and replication protein A is compromised by a cancer-predisposing mutation in BRCA2. Oncogene 22, 28–33 (2003)

    Article  CAS  Google Scholar 

  31. Hartley, C. L. & McCulloch, R. Trypanosoma brucei BRCA2 acts in antigenic variation and has undergone a recent expansion in BRC repeat number that is important during homologous recombination. Mol. Microbiol. 68, 1237–1251 (2008)

    Article  CAS  Google Scholar 

  32. Benson, F. E., Stasiak, A. & West, S. C. Purification and characterization of the human Rad51 protein, an analogue of E. coli RecA. EMBO J. 13, 5764–5771 (1994)

    Article  CAS  Google Scholar 

  33. Sugiyama, T., Zaitseva, E. M. & Kowalczykowski, S. C. A single-stranded DNA-binding protein is needed for efficient presynaptic complex formation by the Saccharomyces cerevisiae Rad51 protein. J. Biol. Chem. 272, 7940–7945 (1997)

    Article  CAS  Google Scholar 

  34. Sigurdsson, S., Trujillo, K., Song, B., Stratton, S. & Sung, P. Basis for avid homologous DNA strand exchange by human Rad51 and RPA. J. Biol. Chem. 276, 8798–8806 (2001)

    Article  CAS  Google Scholar 

  35. Lio, Y. C., Mazin, A. V., Kowalczykowski, S. C. & Chen, D. J. Complex formation by the human Rad51B and Rad51C DNA repair proteins and their activities in vitro . J. Biol. Chem. 278, 2469–2478 (2003)

    Article  CAS  Google Scholar 

  36. Morimatsu, K. & Kowalczykowski, S. C. RecFOR proteins load RecA protein onto gapped DNA to accelerate DNA strand exchange: a universal step of recombinational repair. Mol. Cell 11, 1337–1347 (2003)

    Article  CAS  Google Scholar 

  37. New, J. H., Sugiyama, T., Zaitseva, E. & Kowalczykowski, S. C. Rad52 protein stimulates DNA strand exchange by Rad51 and replication protein A. Nature 391, 407–410 (1998)

    Article  ADS  CAS  Google Scholar 

  38. Shinohara, A. & Ogawa, T. Stimulation by Rad52 of yeast Rad51-mediated recombination. Nature 391, 404–407 (1998)

    Article  ADS  CAS  Google Scholar 

  39. Sung, P. Function of yeast Rad52 protein as a mediator between replication protein A and the Rad51 recombinase. J. Biol. Chem. 272, 28194–28197 (1997)

    Article  CAS  Google Scholar 

  40. 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  Google Scholar 

  41. Sugiyama, T., New, J. H. & Kowalczykowski, S. C. DNA annealing by Rad52 protein is stimulated by specific interaction with the complex of replication protein A and single-stranded DNA. Proc. Natl Acad. Sci. USA 95, 6049–6054 (1998)

    Article  ADS  CAS  Google Scholar 

  42. Kantake, N., Madiraju, M. V., Sugiyama, T. & Kowalczykowski, S. C. Escherichia coli RecO protein anneals ssDNA complexed with its cognate ssDNA-binding protein: a common step in genetic recombination. Proc. Natl Acad. Sci. USA 99, 15327–15332 (2002)

    Article  ADS  CAS  Google Scholar 

  43. Mazloum, N., Zhou, Q. & Holloman, W. K. DNA binding, annealing, and strand exchange activities of Brh2 protein from Ustilago maydis . Biochemistry 46, 7163–7173 (2007)

    Article  CAS  Google Scholar 

  44. Stark, J. M., Pierce, A. J., Oh, J., Pastink, A. & Jasin, M. Genetic steps of mammalian homologous repair with distinct mutagenic consequences. Mol. Cell. Biol. 24, 9305–9316 (2004)

    Article  CAS  Google Scholar 

  45. Liu, J., Doty, T., Gibson, B. & Heyer, W.-D. Human BRCA2 protein promotes RAD51 filament formation on RPA-covered ssDNA. Nature Struct. Mol. Biol. (in the press)

  46. 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  ADS  CAS  Google Scholar 

  47. 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  Google Scholar 

  48. Miné, J. et al. Real-time measurements of the nucleation, growth and dissociation of single Rad51-DNA nucleoprotein filaments. Nucleic Acids Res. 35, 7171–7187 (2007)

    Article  Google Scholar 

  49. Baumann, P. & West, S. C. Heteroduplex formation by human Rad51 protein: effects of DNA end-structure, hRP-A and hRad52. J. Mol. Biol. 291, 363–374 (1999)

    Article  CAS  Google Scholar 

  50. Tutt, A. et al. Mutation in Brca2 stimulates error-prone homology-directed repair of DNA double-strand breaks occurring between repeated sequences. EMBO J. 20, 4704–4716 (2001)

    Article  CAS  Google Scholar 

  51. LeBowitz, J. H. Biochemical Mechanism of Strand Initiation in Bacteriophage Lambda DNA Replication. Ph.D. thesis, Johns Hopkins Univ. (1985)

    Google Scholar 

  52. Mirshad, J. K. & Kowalczykowski, S. C. Biochemical characterization of a mutant RecA protein altered in DNA-binding loop 1. Biochemistry 42, 5945–5954 (2003)

    Article  CAS  Google Scholar 

  53. Zaitseva, E. M., Zaitsev, E. N. & Kowalczykowski, S. C. The DNA binding properties of Saccharomyces cerevisiae Rad51 protein. J. Biol. Chem. 274, 2907–2915 (1999)

    Article  CAS  Google Scholar 

Download references

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.

Author information

Authors and Affiliations

Authors

Contributions

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.

Corresponding author

Correspondence to Stephen C. Kowalczykowski.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Figures

This file contains Supplementary Figures 1-12 with legends. (PDF 3807 kb)

PowerPoint slides

Rights and permissions

Reprints and permissions

About this article

Cite this article

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

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature09399

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Quick links

Nature Briefing: Cancer

Sign up for the Nature Briefing: Cancer newsletter — what matters in cancer research, free to your inbox weekly.

Get what matters in cancer research, free to your inbox weekly. Sign up for Nature Briefing: Cancer