Identification of Holliday junction resolvases from humans and yeast

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Four-way DNA intermediates, also known as Holliday junctions, are formed during homologous recombination and DNA repair, and their resolution is necessary for proper chromosome segregation. Here we identify nucleases from Saccharomyces cerevisiae and human cells that promote Holliday junction resolution, in a manner analogous to that shown by the Escherichia coli Holliday junction resolvase RuvC. The human Holliday junction resolvase, GEN1, and its yeast orthologue, Yen1, were independently identified using two distinct experimental approaches: GEN1 was identified by mass spectrometry following extensive fractionation of HeLa cell-free extracts, whereas Yen1 was detected by screening a yeast gene fusion library for nucleases capable of Holliday junction resolution. The eukaryotic Holliday junction resolvases represent a new subclass of the Rad2/XPG family of nucleases. Recombinant GEN1 and Yen1 resolve Holliday junctions by the introduction of symmetrically related cuts across the junction point, to produce nicked duplex products in which the nicks can be readily ligated.

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Figure 1: Identification of human Holliday junction resolvase activity.
Figure 2: Yeast screen for Holliday junction resolution activities.
Figure 3: Resolution of Holliday junctions by recombinant Yen1 and GEN1.
Figure 4: Specificity of GEN1 Holliday junction resolvase.


  1. 1

    Holliday, R. A mechanism for gene conversion in fungi. Genet. Res. Camb. 5, 282–304 (1964)

  2. 2

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

  3. 3

    Lilley, D. M. J. & White, M. F. The junction-resolving enzymes. Nature Rev. Mol. Cell Biol. 2, 433–443 (2001)

  4. 4

    West, S. C. Processing of recombination intermediates by the RuvABC proteins. Annu. Rev. Genet. 31, 213–244 (1997)

  5. 5

    Kleff, S., Kemper, B. & Sternglanz, R. Identification and characterization of yeast mutants and the gene for a cruciform cutting endonuclease. EMBO J. 11, 699–704 (1992)

  6. 6

    Whitby, M. C. & Dixon, J. Substrate specificity of the SpCCE1 Holliday junction resolvase of Schizosaccharomyces pombe . J. Biol. Chem. 273, 35063–35073 (1998)

  7. 7

    Elborough, K. M. & West, S. C. Resolution of synthetic Holliday junctions in DNA by an endonuclease activity from calf thymus. EMBO J. 9, 2931–2936 (1990)

  8. 8

    Hyde, H., Davies, A. A., Benson, F. E. & West, S. C. Resolution of recombination intermediates by a mammalian endonuclease activity functionally analogous to Escherichia coli RuvC resolvase. J. Biol. Chem. 269, 5202–5209 (1994)

  9. 9

    Constantinou, A., Davies, A. A. & West, S. C. Branch migration and Holliday junction resolution catalyzed by activities from mammalian cells. Cell 104, 259–268 (2001)

  10. 10

    Constantinou, A., Chen, X.-B., McGowan, C. H. & West, S. C. Holliday junction resolution in human cells: Two junction endonucleases with distinct substrate specificities. EMBO J. 21, 5577–5585 (2002)

  11. 11

    Liu, Y., Masson, J.-Y., Shah, R., O’Regan, P. & West, S. C. RAD51C is required for Holliday junction processing in mammalian cells. Science 303, 243–246 (2004)

  12. 12

    Wu, L. & Hickson, I. D. The Bloom’s syndrome helicase suppresses crossing over during homologous recombination. Nature 426, 870–874 (2003)

  13. 13

    Chen, X. B. et al. Human MUS81-associated endonuclease cleaves Holliday junctions in vitro . Mol. Cell 8, 1117–1127 (2001)

  14. 14

    Ciccia, A., Constantinou, A. & West, S. C. Identification and characterization of the human MUS81–EME1 endonuclease. J. Biol. Chem. 278, 25172–25178 (2003)

  15. 15

    Taylor, E. R. & McGowan, C. H. Cleavage mechanism of human MUS81–EME1 acting on Holliday-junction structures. Proc. Natl Acad. Sci. USA 105, 3757–3762 (2008)

  16. 16

    Ghaemmaghami, S. et al. Global analysis of protein expression in yeast. Nature 425, 737–741 (2003)

  17. 17

    Fricke, W. M. & Brill, S. J. Slx1-Slx4 is a second structure-specific endonuclease functionally redundant with Sgs1-Top3. Genes Dev. 17, 1768–1778 (2003)

  18. 18

    Lieber, M. R. The FEN-1 family of structure-specific nucleases in eukaryotic DNA replication, recombination and repair. Bioessays 19, 233–240 (1997)

  19. 19

    Shen, B., Nolan, J. P., Sklar, L. A. & Park, M. S. Functional analysis of point mutations in human flap endonuclease-I active site. Nucleic Acids Res. 25, 3332–3338 (1997)

  20. 20

    Tran, P. T., Erdeniz, N., Dudley, S. & Liskay, R. M. Characterization of nuclease-dependent functions of Exo1p in Saccharomyces cerevisiae . DNA Repair 1, 895–912 (2002)

  21. 21

    Dunderdale, H. J. et al. Formation and resolution of recombination intermediates by E. coli RecA and RuvC proteins. Nature 354, 506–510 (1991)

  22. 22

    Iwasaki, H., Takahagi, M., Shiba, T., Nakata, A. & Shinagawa, H. Escherichia coli RuvC protein is an endonuclease that resolves the Holliday structure. EMBO J. 10, 4381–4389 (1991)

  23. 23

    Boddy, M. N. et al. Mus81-Eme1 are essential components of a Holliday junction resolvase. Cell 107, 537–548 (2001)

  24. 24

    O’Donovan, A., Davies, A. A., Moggs, J. G., West, S. C. & Wood, R. D. XPG endonuclease makes the 3′ incision in human DNA nucleotide excision repair. Nature 371, 432–435 (1994)

  25. 25

    Liu, Y., Kao, H. I. & Bambara, R. A. Flap endonuclease I: a central component of DNA metabolism. Annu. Rev. Biochem. 73, 589–615 (2004)

  26. 26

    Tran, P. T., Erdeniz, N., Symington, L. S. & Liskay, R. M. EXO1 - A multi-tasking eukaryotic nuclease. DNA Repair 3, 1549–1559 (2004)

  27. 27

    Smith, G. R., Boddy, M. N., Shanahan, P. & Russell, P. Fission yeast Mus81-Eme1 Holliday junction resolvase is required for meiotic crossing over but not for gene conversion. Genetics 165, 2289–2293 (2003)

  28. 28

    Osman, F., Dixon, J., Doe, C. L. & Whitby, M. C. Generating crossovers by resolution of nicked Holliday junctions: A role of Mus81-Eme1 in meiosis. Mol. Cell 12, 761–774 (2003)

  29. 29

    de los Santos, T. et al. The Mus81-Mms4 endonuclease acts independently of double-Holliday junction resolution to promote a distinct subset of crossovers during meiosis in budding yeast. Genetics 164, 81–94 (2003)

  30. 30

    Dendouga, N. et al. Disruption of murine Mus81 increases genomic instability and DNA damage sensitivity but does not promote tumorigenesis. Mol. Cell. Biol. 25, 7569–7579 (2005)

  31. 31

    McPherson, J. P. et al. Involvement of mammalian Mus81 in genome integrity and tumor suppression. Science 304, 1822–1826 (2004)

  32. 32

    Oh, S. D., Lao, J. P., Taylor, A. F., Smith, G. R. & Hunter, N. RecQ helicase, Sgs1, and XPF family endonuclease, Mus81-Mms4, resolve aberrant joint molecules during meiotic recombination. Mol. Cell 31, 324–336 (2008)

  33. 33

    Jessop, L. & Lichten, M. Mus81/Mms4 endonuclease and Sgs1 helicase collaborate to ensure proper recombination intermediate metabolism during meiosis. Mol. Cell 31, 313–323 (2008)

  34. 34

    Furukawa, F. et al. OsSEND-1: a new Rad2 nuclease family member in higher plants. Plant Mol. Biol. 51, 59–70 (2003)

  35. 35

    Moritoh, S. et al. RNAi-mediated silencing of OsGEN-L (OsGEN-like), a new member of the Rad2/XPG nuclease family, causes male sterility by defect of microspore development in rice. Plant Cell Physiol. 46, 699–715 (2005)

  36. 36

    Ishikawa, G. et al. DmGEN, a novel Rad2 family endo-exonuclease from Drosophila melanogaster . Nucleic Acids Res. 32, 6251–6259 (2004)

  37. 37

    Kanai, Y. et al. DmGEN shows a flap endonuclease activity, cleaving the blocked-flap structure and model replication fork. FEBS J. 274, 3914–3927 (2007)

  38. 38

    Wood, L. D. et al. The genomic landscapes of human breast and colorectal cancers. Science 318, 1108–1113 (2007)

  39. 39

    Rass, U. & West, S. C. Synthetic junctions as tools to identify and characterise Holliday junction resolvases. Methods Enzymol. 408, 485–501 (2006)

  40. 40

    Eggleston, A. K., Mitchell, A. H. & West, S. C. In vitro reconstitution of the late steps of genetic recombination in E. coli . Cell 89, 607–617 (1997)

  41. 41

    Letunic, I. et al. SMART 5: domains in the context of genomes and networks. Nucleic Acids Res. 34, D257–D260 (2006)

  42. 42

    Larkin, M. A. et al. Clustal W and Clustal X version 2.0. Bioinformatics 23, 2947–2948 (2007)

  43. 43

    Page, R. D. M. TreeView: An application to display phylogenic trees on personal computers. Bioinformatics 12, 357–358 (1996)

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We thank M. Lichten and J. Haber for communication of unpublished work, J.-Y. Bleuyard, A. Deans and other members of our laboratory, past and present, for their input and discussions. We acknowledge the contributions of K. Elborough, H. Hyde, B. Kysela, A. Davies, A. Constantinou and Y. Liu for early studies that underpin this work, which was supported by Cancer Research UK, the Louis-Jeantet Foundation and the EU DNA Repair Consortium. S.C.Y.I. was supported by the Croucher Foundation. M.G.B. is on leave from the University of Santiago de Compostela, and supported by fellowships from the Xunta de Galicia and the Spanish Ministry of Education.

Author Contributions S.C.Y.I. identified ResA as GEN1 and characterized the recombinant protein. U.R. designed and carried out the yeast screen to identify Yen1. M.G.B. expressed and characterized recombinant Yen1. H.R.F. carried out mass spectrometric analyses and together with J.M.S. analysed the MS data. S.C.W. helped with experimental design and wrote the manuscript.

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Correspondence to Stephen C. West.

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Ip, S., Rass, U., Blanco, M. et al. Identification of Holliday junction resolvases from humans and yeast. Nature 456, 357–361 (2008) doi:10.1038/nature07470

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