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Interaction with the BRCA2 C terminus protects RAD51–DNA filaments from disassembly by BRC repeats

A Corrigendum to this article was published on 01 July 2007

Abstract

BRCA2 has an essential function in DNA repair by homologous recombination, interacting with RAD51 via short motifs in the middle and at the C terminus of BRCA2. Here, we report that a conserved 36-residue sequence of human BRCA2 encoded by exon 27 (BRCA2Exon27) interacts with RAD51 through the specific recognition of oligomerized RAD51 ATPase domains. BRCA2Exon27 binding stabilizes the RAD51 nucleoprotein filament against disassembly by BRC repeat 4. The protection is specific for RAD51 filaments formed on single-stranded DNA and is lost when BRCA2Exon27 is phosphorylated on Ser3291. We propose that productive recombination results from the functional balance between the different RAD51-binding modes of the BRC repeat and exon 27 regions of BRCA2. Our results further suggest a mechanism in which CDK phosphorylation of BRCA2Exon27 at the G2-M transition alters the balance in favor of RAD51 filament disassembly, thus terminating recombination.

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Figure 1: Structure and function of BRCA2 and RAD51 proteins.
Figure 2: BRCA2Exon27 binds oligomeric RAD51.
Figure 3: Cross-linking of RAD51, RAD51–BRC4 and RAD51–BRCA2Exon27 complexes.
Figure 4: BRCA2Exon27 binding requires an oligomeric RAD51 assembly.
Figure 5: BRCA2Exon27 binds RAD51 nucleoprotein filaments.
Figure 6: BRCA2Exon27 protects RAD51 nucleoprotein filaments.
Figure 7: EM of RAD51 nucleoprotein filaments.
Figure 8: BRCA2Exon27 protection is specific for ssDNA and independent of ATP hydrolysis.

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References

  1. Rahman, N. & Stratton, M. The genetics of breast cancer susceptibility. Annu. Rev. Genet. 32, 95–121 (1998).

    Article  CAS  Google Scholar 

  2. Nathanson, K.L., Wooster, R., Weber, B.L. & Nathanson, K.N. Breast cancer genetics: what we know and what we need. Nat. Med. 7, 552–556 (2001).

    Article  CAS  Google Scholar 

  3. Connor, F. et al. Tumorigenesis and a DNA repair defect in mice with a truncating BRCA2 mutation. Nat. Genet. 17, 423–430 (1997).

    Article  CAS  Google Scholar 

  4. Patel, K.J. et al. Involvement of BRCA2 in DNA repair. Mol. Cell 1, 347–357 (1998).

    Article  CAS  Google Scholar 

  5. 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 

  6. 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 

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

    Article  CAS  Google Scholar 

  8. 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 G(2)/M checkpoint control. J. Biol. Chem. 274, 32931–32935 (1999).

    Article  CAS  Google Scholar 

  9. 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 

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

    Article  CAS  Google Scholar 

  11. Shivji, M.K. et al. A region of human BRCA2 containing multiple BRC repeats promotes RAD51-mediated strand exchange. Nucleic Acids Res. 34, 4000–4011 (2006).

    Article  CAS  Google Scholar 

  12. 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  CAS  Google Scholar 

  13. Yu, D.S. et al. Dynamic control of Rad51 recombinase by self-association and interaction with BRCA2. Mol. Cell 12, 1029–1041 (2003).

    Article  CAS  Google Scholar 

  14. Pellegrini, L. & Venkitaraman, A. Emerging functions of BRCA2 in DNA recombination. Trends Biochem. Sci. 29, 310–316 (2004).

    Article  CAS  Google Scholar 

  15. 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  CAS  Google Scholar 

  16. 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 

  17. Wong, A.K., 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 

  18. Mizuta, R. et al. RAB22 and RAB163/mouse BRCA2: proteins that specifically interact with the RAD51 protein. Proc. Natl. Acad. Sci. USA 94, 6927–6932 (1997).

    Article  CAS  Google Scholar 

  19. Shin, D.S. et al. Full-length archaeal Rad51 structure and mutants: mechanisms for RAD51 assembly and control by BRCA2. EMBO J. 22, 4566–4576 (2003).

    Article  CAS  Google Scholar 

  20. Kinebuchi, T. et al. Structural basis for octameric ring formation and DNA interaction of the human homologous-pairing protein dmc1. Mol. Cell 14, 363–374 (2004).

    Article  CAS  Google Scholar 

  21. Conway, A.B. et al. Crystal structure of a Rad51 filament. Nat. Struct. Mol. Biol. 11, 791–796 (2004).

    Article  CAS  Google Scholar 

  22. Wu, Y., Qian, X., He, Y., Moya, I.A. & Luo, Y. Crystal structure of an ATPase-active form of Rad51 homolog from methanococcus voltae: insights into potassium-dependence. J. Biol. Chem. 280, 722–728 (2004).

    Article  Google Scholar 

  23. Wu, Y., He, Y., Moya, I.A., Qian, X. & Luo, Y. Crystal structure of archaeal recombinase RadA: a snapshot of its extended conformation. Mol. Cell 15, 423–435 (2004).

    Article  CAS  Google Scholar 

  24. Wyman, C. & Kanaar, R. Homologous recombination: down to the wire. Curr. Biol. 14, R629–R631 (2004).

    Article  CAS  Google Scholar 

  25. Story, R.M., Weber, I.T. & Steitz, T.A. The structure of the E. coli recA protein monomer and polymer. Nature 355, 318–325 (1992).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  27. Galkin, V.E. et al. BRCA2 BRC motifs bind RAD51-DNA filaments. Proc. Natl. Acad. Sci. USA 102, 8537–8542 (2005).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  29. Donoho, G. et al. Deletion of Brca2 exon 27 causes hypersensitivity to DNA crosslinks, chromosomal instability, and reduced life span in mice. Genes Chromosom. Cancer 36, 317–331 (2003).

    Article  CAS  Google Scholar 

  30. McAllister, K.A. et al. Cancer susceptibility of mice with a homozygous deletion in the COOH-terminal domain of the Brca2 gene. Cancer Res. 62, 990–994 (2002).

    CAS  PubMed  Google Scholar 

  31. Atanassov, B.S., Barrett, J.C. & Davis, B.J. Homozygous germ line mutation in exon 27 of murine Brca2 disrupts the Fancd2-Brca2 pathway in the homologous recombination-mediated DNA interstrand cross-links' repair but does not affect meiosis. Genes Chromosom. Cancer 44, 429–437 (2005).

    Article  CAS  Google Scholar 

  32. Fortin, G.S. & Symington, L.S. Mutations in yeast Rad51 that partially bypass the requirement for Rad55 and Rad57 in DNA repair by increasing the stability of Rad51-DNA complexes. EMBO J. 21, 3160–3170 (2002).

    Article  CAS  Google Scholar 

  33. Tombline, G. & Fishel, R. Biochemical characterization of the human RAD51 protein. I. ATP hydrolysis. J. Biol. Chem. 277, 14417–14425 (2002).

    Article  CAS  Google Scholar 

  34. Baumann, P., Benson, F.E., Hajibagheri, N. & West, S.C. Purification of human Rad51 protein by selective spermidine precipitation. Mutat. Res. 384, 65–72 (1997).

    Article  CAS  Google Scholar 

  35. Peranen, J., Rikkonen, M., Hyvonen, M. & Kaariainen, L. T7 vectors with modified T7lac promoter for the expression of proteins in E. coli. Anal. Biochem. 236, 371–373 (1996).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank T. Blundell, S. Klinge, M. Shivji, E. Rajendra, N. Ayoub and A. Venkitaraman for comments and advice, N. Allcott and T. Yamamoto for their contributions in the early stages of this work, and J. Skepper (University of Cambridge) for assistance with EM. This work was supported by a Wellcome Trust Senior Fellowship in basic biomedical sciences to L.P. and a Biotechnology and Biological Sciences Research Council PhD studentship to O.R.D.

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O.R.D. and L.P. designed the experiments and interpreted the data; O.R.D. performed all experiments.

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Correspondence to Luca Pellegrini.

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The authors declare no competing financial interests.

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Preparation of DNA substrates. (PDF 43 kb)

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Davies, O., Pellegrini, L. Interaction with the BRCA2 C terminus protects RAD51–DNA filaments from disassembly by BRC repeats. Nat Struct Mol Biol 14, 475–483 (2007). https://doi.org/10.1038/nsmb1251

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