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Identification of Holliday junction resolvases from humans and yeast

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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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We thank M. Lichten and J. Haber for communication of unpublished work, J.-Y. Bleuyard, A. Deans and other members of our laboratory, past and present, for their input and discussions.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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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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