An In Vitro Assay for Monitoring the Formation and Branch Migration of Holliday Junctions Mediated by a Eukaryotic Recombinase

  • Yasuto Murayama
  • Hiroshi IwasakiEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 745)


DNA strand exchange is a core reaction of homologous recombination directly catalyzed by Rad51/Dmc1 RecA family recombinases in eukaryotes. This reaction proceeds through the formation of several DNA intermediates. The X-shaped four-way DNA structure known as a Holliday junction (HJ) is a central intermediate in homologous recombination. Genetic and biochemical studies indicate that the HJ is important for the production of crossover-type recombinants, which are reciprocal exchange products. According to a recombination model for the repair of DNA double-strand breaks, the formation of HJs requires a reciprocal duplex–duplex DNA exchange known as the DNA four-strand exchange reaction. In vitro analyses using purified recombination proteins and model DNA substrates provide a mechanistic insight into the DNA strand exchange reaction, including the steps leading to the formation and branch migration of Holliday junctions.

Key words

Homologous recombination Holliday junction Rad51 recombinase DNA strand exchange gel electrophoresis 



We thank K. Ito and T. Koizumi for preparation of the manuscript. This study was supported in part by grants in aid for Scientific Research on Priority Areas from the Ministry of Education, Culture, Sports, Science, and Technology (MECSST) of Japan and for scientific research (A) to HI and for young scientist (B) to YM from the Japan Society for the Promotion of Science (JSPS), and by the Uehara Memorial Foundation to HI.


  1. 1.
    Masson, J.Y., and West, S.C. (2001) The Rad51 and Dmc1 recombinases: a non-identical twin relationship. Trends Biochem Sci 26, 131–136.PubMedCrossRefGoogle Scholar
  2. 2.
    Neale, M.J., and Keeney, S. (2006) Clarifying the mechanics of DNA strand exchange in meiotic recombination. Nature 442, 153–158.PubMedCrossRefGoogle Scholar
  3. 3.
    Cox, M.M. (2007) Motoring along with the bacterial RecA protein. Nat Rev Mol Cell Biol 8, 127–138.PubMedCrossRefGoogle Scholar
  4. 4.
    Haruta, N., Akamatsu, Y., Tsutsui, Y., Kurokawa, Y., Murayama, Y., Arcangioli, B., and Iwasaki, H. (2008) Fission yeast Swi5 protein, a novel DNA recombination mediator. DNA Repair (Amst) 7, 1–9.CrossRefGoogle Scholar
  5. 5.
    Sung, P., and Klein, H. (2006) Mechanism of homologous recombination: mediators and helicases take on regulatory functions. Nat Rev Mol Cell Biol 7, 739–750.PubMedCrossRefGoogle Scholar
  6. 6.
    Sugiyama, T., Zaitseva, E.M., and Kowalczykowski, S.C. (1997) 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.PubMedCrossRefGoogle Scholar
  7. 7.
    Symington, L.S. (2002) Role of RAD52 epistasis group genes in homologous recombination and double-strand break repair. Microbiol Mol Biol Rev 66, 630–670.PubMedCrossRefGoogle Scholar
  8. 8.
    Murayama, Y., Kurokawa, Y., Mayanagi, K., and Iwasaki, H. (2008) Formation and branch migration of Holliday junctions mediated by eukaryotic recombinases. Nature 451, 1018–1021.PubMedCrossRefGoogle Scholar
  9. 9.
    Haruta, N., Kurokawa, Y., Murayama, Y., Akamatsu, Y., Unzai, S., Tsutsui, Y., and Iwasaki, H. (2006) The Swi5-Sfr1 complex stimulates Rhp51/Rad51- and Dmc1-mediated DNA strand exchange in vitro. Nat Struct Mol Biol 13, 823–830.PubMedCrossRefGoogle Scholar
  10. 10.
    Muris, D.F., Vreeken, K., Carr, A.M., Broughton, B.C., Lehmann, A.R., Lohman, P.H., and Pastink, A. (1993) Cloning the RAD51 homologue of Schizosaccharomyces pombe. Nucleic Acids Res 21, 4586–4591.PubMedCrossRefGoogle Scholar
  11. 11.
    Akamatsu, Y., Dziadkowiec, D., Ikeguchi, M., Shinagawa, H., and Iwasaki, H. (2003) Two different Swi5-containing protein complexes are involved in mating-type switching and recombination repair in fission yeast. Proc Natl Acad Sci USA 100, 15770–15775.PubMedCrossRefGoogle Scholar
  12. 12.
    Kurokawa, Y., Murayama, Y., Haruta-Takahashi, N., Urabe, I., and Iwasaki, H. (2008) Reconstitution of DNA strand exchange mediated by Rhp51 recombinase and two mediators. PLoS Biol 6, e88.PubMedCrossRefGoogle Scholar
  13. 13.
    Connolly, B., Parsons, C.A., Benson, F.E., Dunderdale, H.J., Sharples, G.J., Lloyd, R.G., and West, S.C. (1991) Resolution of Holliday junctions in vitro requires the Escherichia coli ruvC gene product. Proc Natl Acad Sci USA 88, 6063–6067.PubMedCrossRefGoogle Scholar
  14. 14.
    Iwasaki, H., Takahagi, M., Shiba, T., Nakata, A., and Shinagawa, H. (1991) Escherichia coli RuvC protein is an endonuclease that resolves the Holliday structure. EMBO J 10, 4381–4389.PubMedGoogle Scholar
  15. 15.
    West, S.C., Cassuto, E., and Howard-Flanders, P. (1982) Postreplication repair in E. coli: strand exchange reactions of gapped DNA by RecA protein. Mol Gen Genet 187, 209–217.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  1. 1.Cancer Research UK, London Research InstituteLondonUK
  2. 2.School and Graduate School of Bioscience and Biotechnology, Tokyo Institute of TechnologyTokyoJapan

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