Article | Published:

Rad54 dissociates homologous recombination intermediates by branch migration

Nature Structural & Molecular Biology volume 14, pages 746753 (2007) | Download Citation

Abstract

Double-strand DNA breaks (DSBs) cause cell death and genome instability. Homologous recombination is a major DSB repair pathway that operates by forming joint molecules with homologous DNA sequences, which are used as templates to achieve accurate repair. In eukaryotes, Rad51 protein (RecA homolog) searches for homologous sequences and catalyzes the formation of joint molecules (D-loops). Once joint molecules have been formed, DNA polymerase extends the 3′ single-stranded DNA tails of the broken chromosome, restoring the lost information. How joint molecules subsequently dissociate is unknown. We reconstituted DSB repair in vitro using purified human homologous recombination proteins and DNA polymerase η. We found that Rad54 protein, owing to its ATP-dependent branch-migration activity, can cause dissociation of joint molecules. These results suggest a previously uncharacterized mechanism of DSB repair in which Rad54 branch-migration activity plays an important role.

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References

  1. 1.

    & Mechanism of homologous recombination: mediators and helicases take on regulatory functions. Nat. Rev. Mol. Cell Biol. 7, 739–750 (2006).

  2. 2.

    Making crossovers during meiosis. Biochem. Soc. Trans. 33, 1451–1455 (2005).

  3. 3.

    & Recombination proteins in yeast. Annu. Rev. Genet. 38, 233–271 (2004).

  4. 4.

    , & DNA double-strand break repair and chromosome translocations. DNA Repair (Amst.) 5, 1075–1081 (2006).

  5. 5.

    & Happy Hollidays: 40th anniversary of the Holliday junction. Nat. Rev. Mol. Cell Biol. 5, 937–944 (2004).

  6. 6.

    , & DNA strand exchange proteins: a biochemical and physical comparison. Front. Biosci. 3, D570–D603 (1998).

  7. 7.

    , , & Rad51 recombinase and recombination mediators. J. Biol. Chem. 278, 42729–42732 (2003).

  8. 8.

    & Differential timing and control of noncrossover and crossover recombination during meiosis. Cell 106, 47–57 (2001).

  9. 9.

    & The single-end invasion: an asymmetric intermediate at the double-strand break to double-holliday junction transition of meiotic recombination. Cell 106, 59–70 (2001).

  10. 10.

    et al. Single Holliday junctions are intermediates of meiotic recombination. Cell 127, 1167–1178 (2006).

  11. 11.

    & Identification of double Holliday junctions as intermediates in meiotic recombination. Cell 83, 783–791 (1995).

  12. 12.

    & Multiple pathways of recombination induced by double-strand breaks in Saccharomyces cerevisiae. Microbiol. Mol. Biol. Rev. 63, 349–404 (1999).

  13. 13.

    , , & Rad54: the Swiss Army knife of homologous recombination? Nucleic Acids Res. 34, 4115–4125 (2006).

  14. 14.

    , & Rad54 protein promotes branch migration of Holliday junctions. Nature 442, 590–593 (2006).

  15. 15.

    et al. Structure of the SWI2/SNF2 chromatin-remodeling domain of eukaryotic Rad54. Nat. Struct. Mol. Biol. 12, 350–356 (2005).

  16. 16.

    , & Rad54, a Jack of all trades in homologous recombination. DNA Repair (Amst.) 2, 787–794 (2003).

  17. 17.

    , & Catalysis of homologous DNA pairing by yeast Rad51 and Rad54 proteins. Nature 393, 91–94 (1998).

  18. 18.

    & Human Rad54 protein stimulates DNA strand exchange activity of hRad51 protein in the presence of Ca2+. J. Biol. Chem. 279, 52042–52051 (2004).

  19. 19.

    , , , & Rad54 protein stimulates heteroduplex DNA formation in the synaptic phase of DNA strand exchange via specific interactions with the presynaptic Rad51 nucleoprotein filament. J. Mol. Biol. 307, 1207–1221 (2001).

  20. 20.

    , , , & Superhelicity-driven homologous DNA pairing by yeast recombination factors Rad51 and Rad54. Mol. Cell 6, 563–572 (2000).

  21. 21.

    , , & The architecture of the human Rad54-DNA complex provides evidence for protein translocation along DNA. Proc. Natl. Acad. Sci. USA 98, 8454–8460 (2001).

  22. 22.

    , & Visualization of Rad54, a chromatin remodeling protein, translocating on single DNA molecules. Mol. Cell 23, 143–148 (2006).

  23. 23.

    , & Rad54, a Swi2/Snf2-like Recombinational Repair Protein, Disassembles Rad51:dsDNA Filaments. Mol. Cell 10, 1175–1188 (2002).

  24. 24.

    , & Rad54 protein possesses chromatin-remodeling activity stimulated by the Rad51-ssDNA nucleoprotein filament. Nat. Struct. Biol. 10, 182–186 (2003).

  25. 25.

    & Strand pairing by Rad54 and Rad51 is enhanced by chromatin. Genes Dev. 16, 2767–2771 (2002).

  26. 26.

    , , , & Rad54p is a chromatin remodeling enzyme required for heteroduplex DNA joint formation with chromatin. J. Biol. Chem. 278, 9212–9218 (2003).

  27. 27.

    , , , & Spontaneous and double-strand break-induced recombination, and gene conversion tract lengths, are differentially affected by overexpression of wild-type or ATPase-defective yeast Rad54. Nucleic Acids Res. 30, 2727–2735 (2002).

  28. 28.

    & Chromatin remodeling by ATP-dependent molecular machines. Bioessays 25, 1192–1200 (2003).

  29. 29.

    , & Promotion of Rad51-dependent D-loop formation by yeast recombination factor Rdh54/Tid1. Genes Dev. 14, 2206–2215 (2000).

  30. 30.

    et al. Binding and melting of D-loops by the Bloom syndrome helicase. Biochemistry 39, 14617–14625 (2000).

  31. 31.

    , & Holliday junction-binding peptides inhibit distinct junction-processing enzymes. Proc. Natl. Acad. Sci. USA 102, 6867–6872 (2005).

  32. 32.

    & Ca2+ activates human homologous recombination protein Rad51 by modulating its ATPase activity. Proc. Natl. Acad. Sci. USA 101, 9988–9993 (2004).

  33. 33.

    , , , & Roles of ATP binding and ATP hydrolysis in human Rad51 recombinase function. DNA Repair (Amst.) 5, 381–391 (2006).

  34. 34.

    , & The requirement for ATP hydrolysis by Saccharomyces cerevisiae Rad51 is bypassed by mating-type heterozygosity or RAD54 in high copy. Mol. Cell. Biol. 22, 6336–6343 (2002).

  35. 35.

    , , & The rad51–K191R ATPase-defective mutant is impaired for presynaptic filament formation. Mol. Cell. Biol. 26, 9544–9554 (2006).

  36. 36.

    , , & Rad52-mediated DNA annealing after Rad51-mediated DNA strand exchange promotes second ssDNA capture. EMBO J. 25, 5539–5548 (2006).

  37. 37.

    et al. Dual roles for DNA polymerase eta in homologous DNA recombination and translesion DNA synthesis. Mol. Cell 20, 793–799 (2005).

  38. 38.

    et al. Human DNA polymerase eta promotes DNA synthesis from strand invasion intermediates of homologous recombination. Mol. Cell 20, 783–792 (2005).

  39. 39.

    , & In vivo roles of Rad52, Rad54, and Rad55 proteins in Rad51-mediated recombination. Mol. Cell 12, 209–219 (2003).

  40. 40.

    , , & Recruitment of the recombinational repair machinery to a DNA double-strand break in yeast. Mol. Cell 12, 221–232 (2003).

  41. 41.

    , , , & In vivo assembly and disassembly of Rad51 and Rad52 complexes during double-strand break repair. EMBO J. 23, 939–949 (2004).

  42. 42.

    , , & CeBRC-2 stimulates D-loop formation by RAD-51 and promotes DNA single-strand annealing. J. Mol. Biol. 361, 231–242 (2006).

  43. 43.

    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).

  44. 44.

    et al. Direct interaction of FANCD2 with BRCA2 in DNA damage response pathways. Hum. Mol. Genet. 13, 1241–1248 (2004).

  45. 45.

    , , , & Saccharomyces cerevisiae Mer3 helicase stimulates 3′-5′ heteroduplex extension by Rad51; implications for crossover control in meiotic recombination. Cell 117, 47–56 (2004).

  46. 46.

    et al. Differential contributions of mammalian Rad54 paralogs to recombination, DNA damage repair, and meiosis. Mol. Cell. Biol. 26, 976–989 (2006).

  47. 47.

    , & Mobile D-loops are a preferred substrate for the Bloom's syndrome helicase. Nucleic Acids Res. 34, 2269–2279 (2006).

  48. 48.

    RecQ helicases: caretakers of the genome. Nat. Rev. Cancer 3, 169–178 (2003).

  49. 49.

    , & Drosophila BLM in double-strand break repair by synthesis-dependent strand annealing. Science 299, 265–267 (2003).

  50. 50.

    & The Bloom's syndrome helicase suppresses crossing over during homologous recombination. Nature 426, 870–874 (2003).

  51. 51.

    Mechanism and control of recombination in fungi. Mutat. Res. 284, 97–110 (1992).

  52. 52.

    , , , & Srs2 and Sgs1-Top3 suppress crossovers during double-strand break repair in yeast. Cell 115, 401–411 (2003).

  53. 53.

    et al. Possible association of BLM in decreasing DNA double strand breaks during DNA replication. EMBO J. 19, 3428–3435 (2000).

  54. 54.

    & Strand exchange activity of human recombination protein Rad52. Proc. Natl. Acad. Sci. USA 101, 9562–9567 (2004).

  55. 55.

    , & Recombinant replication protein A: expression, complex formation, and functional characterization. J. Biol. Chem. 269, 11121–11132 (1994).

  56. 56.

    , , & Mechanisms of accurate translesion synthesis by human DNA polymerase eta. EMBO J. 19, 3100–3109 (2000).

  57. 57.

    , & Analysis of branch migration activities of proteins using synthetic DNA substrates. Nat. Protocols published online 1 September 2006 (doi:10.1038/nprot.2006.217).

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Acknowledgements

We thank P. Sung (Yale University), M. Wold (University of Iowa) and E. Golub (Yale University) for RAD51, RPA and RAD52 expression vectors; G. Schnitzler and N. Ulyanova (Tufts University) for human SWI/SNF protein; Z. Zhang and R. Kingston (Harvard Medical School) for RAD54B protein; and M. Bouchard, M. Rossi and O. Mazina (Drexel University College of Medicine) for comments and discussion. This work was supported by US National Institutes of Health grant CA100839 to A.V.M.

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Affiliations

  1. Department of Biochemistry and Molecular Biology, Drexel University College of Medicine, Philadelphia, Pennsylvania 19102-1192, USA.

    • Dmitry V Bugreev
    •  & Alexander V Mazin
  2. Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of Russian Academy of Science, Novosibirsk, 630090, Russia.

    • Dmitry V Bugreev
  3. Graduate School of Frontier Biosciences, Osaka University and Solution Oriented Research for Science and Technology, Japan Science and Technology Agency, 1-3 Yamada-oka, Suita, Osaka 565-0871, Japan.

    • Fumio Hanaoka

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The authors declare no competing financial interests.

Corresponding author

Correspondence to Alexander V Mazin.

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DOI

https://doi.org/10.1038/nsmb1268

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