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Identification of Holliday junction resolvases from humans and yeast

Nature volume 456, pages 357–361 (2008)Cite this article

Abstract

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.

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References

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

    Article  Google Scholar 

  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)

    CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  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)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  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)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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)

    CAS  PubMed  Google Scholar 

  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)

    Article  CAS  PubMed  Google Scholar 

  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)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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)

    Article  CAS  ADS  PubMed  Google Scholar 

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

    Article  CAS  ADS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  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)

    Article  CAS  ADS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  ADS  PubMed  Google Scholar 

  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)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  MathSciNet  CAS  PubMed  Google Scholar 

  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)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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)

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  ADS  PubMed  Google Scholar 

  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)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  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)

    Article  ADS  PubMed  Google Scholar 

  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)

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  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)

    CAS  PubMed  PubMed Central  Google Scholar 

  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)

    Article  CAS  PubMed  Google Scholar 

  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)

    CAS  PubMed  PubMed Central  Google Scholar 

  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)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  ADS  PubMed  Google Scholar 

  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)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  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)

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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)

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  ADS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  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)

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  Google Scholar 

Download references

Acknowledgements

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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Author notes

  1. Stephen C. Y. Ip, Ulrich Rass and Miguel G. Blanco: These authors contributed equally to this work.

Authors and Affiliations

  1. Genetic Recombination Laboratory,,

    Stephen C. Y. Ip, Ulrich Rass, Miguel G. Blanco & Stephen C. West

  2. Protein Analysis and Proteomics Laboratory, Cancer Research UK, London Research Institute, Clare Hall Laboratories, South Mimms, Herts EN6 3LD, UK ,

    Helen R. Flynn & J. Mark Skehel

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  1. Stephen C. Y. Ip
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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). https://doi.org/10.1038/nature07470

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