Holliday Junction Branch Migration and Resolution Assays

  • Angelos Constantinou
  • Stephen C. West
Part of the Methods in Molecular Biology™ book series (MIMB, volume 262)


Holliday junctions are central intermediates in the process of genetic recombination; they form as a consequence of a reciprocal exchange of strands between paired DNA molecules. Enzymes that specifically recognize and process these junctions are necessary for the formation of recombinant products. In the methods described here, we detail the in vitro construction of two types of Holliday junction: (1) a small synthetic junction formed by the annealing of partially complementary oligonucleotides; and (2) a true recombination intermediate structure formed by RecA protein-mediated strand exchange. The use of these substrates in assays designed to detect Holliday junction branch migration and resolution activities is described.

Key Words

homologous recombination double-strand break repair recombination intermediates RuvABC 


  1. 1.
    Holliday, R. (1964) A mechanism for gene conversion in fungi. Genet. Res. Camb. 5, 282–304.CrossRefGoogle Scholar
  2. 2.
    Seigneur, M., Bidnenko, V., Ehrlich, S. D., and Michel, B. (1998) RuvAB acts at arrested replication forks. Cell 95, 419–430.PubMedCrossRefGoogle Scholar
  3. 3.
    Cox, M. M. (2001) Recombinational DNA repair of damaged replication forks in Escherichia coli: questions. Annu. Rev. Genet. 35, 53–82.PubMedCrossRefGoogle Scholar
  4. 4.
    Sogo, J. M., Lopes, M., and Foiani, M. (2002) Fork reversal and ssDNA accumulation at stalled replication forks owing to checkpoint defects. Science 297, 599–602.PubMedCrossRefGoogle Scholar
  5. 5.
    Postow, L., Ullsperger, C., Keller, R. W., Bustamante, C., Vologodskii, A. V., and Cozzarelli, N. R. (2001) Positive torsional strain causes the formation of a four-way junction at replication forks. J. Biol. Chem. 276, 2790–2796.PubMedCrossRefGoogle Scholar
  6. 6.
    Bolt, E. L. and Lloyd, R. G. (2002) Substrate specificity of RusA resolvase reveals the DNA structures targeted by RuvAB and RecG in vivo. Mol. Cell 10, 187–198.PubMedCrossRefGoogle Scholar
  7. 7.
    Dunderdale, H. J., Benson, F. E., Parsons, C. A., Sharples, G. J., Lloyd, R. G., and West, S. C. (1991) Formation and resolution of recombination intermediates by E. coli RecA and RuvC proteins. Nature 354, 506–510.PubMedCrossRefGoogle Scholar
  8. 8.
    Connolly, B., Parsons, C. A., Benson, F. E., et al. (1991) Resolution of Holliday junctions in vitro requires the Escherichia coli ruvC gene product. Proc. Natl. Acad. Sci. USA 88, 6063–6067.PubMedCrossRefGoogle Scholar
  9. 9.
    Lloyd, R. G. and Sharples, G. J. (1993) Processing of recombination intermediates by the RecG and RuvAB proteins of Escherichia coli. Nucleic Acids Res. 21, 1719–1725.PubMedCrossRefGoogle Scholar
  10. 10.
    Whitby, M. C., Vincent, S. D., and Lloyd, R. G. (1994) Branch migration of Holliday junctions: identification of RecG protein as a junction specific DNA helicase. EMBO J. 13, 5220–5228.PubMedGoogle Scholar
  11. 11.
    Bennett, R. J. and West, S. C. (1996) Resolution of Holliday junctions in genetic recombination: RuvC protein nicks DNA at the point of strand exchange. Proc. Natl. Acad. Sci. USA 93, 12,217–12,222.PubMedCrossRefGoogle Scholar
  12. 12.
    Bennett, R. J. and West, S. C. (1995) Structural analysis of the RuvC-Holliday junction complex reveals an unfolded junction. J. Mol. Biol. 252, 213–226.PubMedCrossRefGoogle Scholar
  13. 13.
    Bennett, R. J., Dunderdale, H. J., and West, S. C. (1993) Resolution of Holliday junctions by RuvC resolvase: cleavage specificity and DNA distortion. Cell 74, 1021–1031.PubMedCrossRefGoogle Scholar
  14. 14.
    Adams, D. E. and West, S. C. (1996) Bypass of DNA heterologies during RuvAB-mediated three-and four-strand branch migration. J. Mol. Biol. 263, 582–596.PubMedCrossRefGoogle Scholar
  15. 15.
    Eggleston, A. K., Mitchell, A. H., and West, S. C. (1997) In vitro reconstitution of the late steps of genetic recombination in E. coli. Cell 89, 607–617.Google Scholar
  16. 16.
    Müller, B., Burdett, I., and West, S. C. (1992) Unusual stability of recombination intermediates made by Escherichia coli RecA protein. EMBO J. 11, 2685–2693.PubMedGoogle Scholar
  17. 17.
    Müller, B., Tsaneva, I. R., and West, S. C. (1993) Branch migration of Holliday junctions promoted by the Escherichia coli RuvA and RuvB proteins: I. Comparison of the RuvAB-and RuvB-mediated reactions. J. Biol. Chem. 268, 17,179–17,184.PubMedGoogle Scholar
  18. 18.
    Müller, B., Tsaneva, I. R., and West, S. C. (1993) Branch migration of Holliday junctions promoted by the Escherichia coli RuvA and RuvB proteins: II. Interaction of RuvB with DNA. J. Biol. Chem. 268, 17,185–17,189.PubMedGoogle Scholar
  19. 19.
    Parsons, C. A., Tsaneva, I., Lloyd, R. G., and West, S. C. (1992) Interaction of Escherichia coli RuvA and RuvB proteins with synthetic Holliday junctions. Proc. Natl. Acad. Sci. USA 89, 5452–5456.PubMedCrossRefGoogle Scholar
  20. 20.
    Parsons, C. A. and West, S. C. (1993) Formation of a RuvAB-Holliday junction complex in vitro. J. Mol. Biol. 232, 397–405.PubMedCrossRefGoogle Scholar
  21. 21.
    Parsons, C. A., Stasiak, A., Bennett, R. J., and West, S. C. (1995) Structure of a multisubunit complex that promotes DNA branch migration. Nature 374, 375–378.PubMedCrossRefGoogle Scholar
  22. 22.
    Shah, R., Bennett, R. J., and West, S. C. (1994) Genetic recombination in E. coli: RuvC protein cleaves Holliday junctions at resolution hotspots in vitro. Cell 79, 853–864.PubMedCrossRefGoogle Scholar
  23. 23.
    Tsaneva, I. R., Müller, B., and West, S. C. (1992) ATP-dependent branch migration of Holliday junctions promoted by the RuvA and RuvB proteins of E. coli. Cell 69, 1171–1180.PubMedCrossRefGoogle Scholar
  24. 24.
    Van Gool, A. J., Hajibagheri, N. M. A., Stasiak, A., and West, S. C. (1999) Assembly of the Escherichia coli RuvABC resolvasome directs the orientation of Holliday junction resolution. Genes Dev. 13, 1861–1870.PubMedCrossRefGoogle Scholar
  25. 25.
    West, S. C. (1997) Processing of recombination intermediates by the RuvABC proteins. Annu. Rev. Genet. 31, 213–244.PubMedCrossRefGoogle Scholar
  26. 26.
    Iwasaki, H., Takahagi, M., Nakata, A., and Shinagawa, H. (1992) Escherichia coli RuvA and RuvB proteins specifically interact with Holliday junctions and promote branch migration. Genes Dev. 6, 2214–2220.PubMedCrossRefGoogle Scholar
  27. 27.
    White, M. F. and Lilley, D. M. J. (1997) The resolving enzyme Cce1 of yeast opens the structure of the 4-way DNA junction. J. Mol. Biol. 266, 122–134.PubMedCrossRefGoogle Scholar
  28. 28.
    White, M. F. and Lilley, D. M. J. (1997) Characterization of a Holliday junction-resolving enzyme from Schizosaccharomyces pombe. Mol. Cell. Biol. 17, 6465–6471.PubMedGoogle Scholar
  29. 29.
    White, M. F. and Lilley, D. M. J. (1998) Interaction of the resolving enzyme Ydc2 with the four-way DNA junction. Nucleic Acids Res. 26, 5609–5616.PubMedCrossRefGoogle Scholar
  30. 30.
    Oram, M., Keeley, A., and Tsaneva, I. (1998) Holliday junction resolvase in Schizosaccharomyces pombe has identical endonuclease activity to the Cce1 homolog Ydc2. Nucleic Acids Res. 26, 594–601.PubMedCrossRefGoogle Scholar
  31. 31.
    Whitby, M. C. and Dixon, J. (1998) Substrate specificity of the Cce1 Holliday junction resolvase of Schizosaccharomyces pombe. J. Biol. Chem. 273, 35,063–35,073.PubMedCrossRefGoogle Scholar
  32. 32.
    Kvaratskhelia, M. and White, M. F. (2000) An archaeal Holliday junction resolving enzyme from Sulfolobus solfataricus exhibits unique properties. J. Mol. Biol. 295, 193–202.PubMedCrossRefGoogle Scholar
  33. 33.
    Kvaratskhelia, M. and White, M. F. (2000) Two Holliday junction resolving enzymes in Sulfolobus solfataricus. J. Mol. Biol. 297, 923–932.PubMedCrossRefGoogle Scholar
  34. 34.
    Bolt, E. L., Lloyd, R. G., and Sharples, G. J. (2001) Genetic analysis of an archaeal Holliday junction resolvase in Escherichia coli. J. Mol. Biol. 310, 577–589.PubMedCrossRefGoogle Scholar
  35. 35.
    Komori, K., Sakae, S., Shinagawa, H., Morikawa, K., and Ishino, Y. (1999) A Holliday junction resolvase from Pyrococcus furiosus: functional similarity to Escherichia coli RuvC provides evidence for conserved mechanism of homologous recombination in Bacteria, Eukarya, and Archaea. Proc. Natl. Acad. Sci. USA 96, 8873–8878.PubMedCrossRefGoogle Scholar
  36. 36.
    Komori, K., Sakae, S., Fujikane, R., Morikawa, K., Shinagawa, H., and Ishino, Y. (2000) Biochemical characterization of the Hjc Holliday junction resolvase of Pyrococcus furiosus. Nucleic Acids Res. 28, 4544–4551.PubMedCrossRefGoogle Scholar
  37. 37.
    Komori, K., Fujikane, R., Shinagawa, H., and Ishino, Y. (2002) Novel endonuclease in Archaea cleaving DNA with various branched structure. Genes Genet. Syst. 77, 227–241.PubMedCrossRefGoogle Scholar
  38. 38.
    Elborough, K. M. and West, S. C. (1990) Resolution of synthetic Holliday junctions in DNA by an endonuclease activity from calf thymus. EMBO J. 9, 2931–2936.PubMedGoogle Scholar
  39. 39.
    Constantinou, A., Tarsounas, M., Karow, J. K., et al. (2000) Werner′s syndrome protein (WRN) migrates Holliday junctions and co-localizes with RPA upon replication arrest. EMBO R. 1, 80–84.CrossRefGoogle Scholar
  40. 40.
    Constantinou, A., Davies, A. A., and West, S. C. (2001) Branch migration and Holliday junction resolution catalyzed by activities from mammalian cells. Cell 104, 259–268.PubMedCrossRefGoogle Scholar
  41. 41.
    Constantinou, A., Chen, X.-B., McGowan, C. H., and West, S. C. (2002) Holliday junction resolution in human cells: two junction endonucleases with distinct substrate specificities. EMBO J. 21, 5577–5585.PubMedCrossRefGoogle Scholar
  42. 42.
    Karow, J. K., Constantinou, A., Li, J.-L., West, S. C., and Hickson, I. D. (2000) The Bloom’s syndrome gene product promotes branch migration of Holliday junctions. Proc. Natl. Acad. Sci. USA 97, 6504–6508.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc. 2004

Authors and Affiliations

  • Angelos Constantinou
    • 1
  • Stephen C. West
    • 2
  1. 1.Institute of BiochemistryUniversity of LausanneEpalingesSwitzerland
  2. 2.London Research Institute, Clare Hall LaboratoriesCancer Research UKUK

Personalised recommendations