Identification of Holliday junction resolvases from humans and yeast.
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.
Ip
Stephen C Y
SC
Genetic Recombination Laboratory, Cancer Research UK, London Research Institute, Clare Hall Laboratories, South Mimms, Herts EN6 3LD, UK.
Rass
Ulrich
U
Blanco
Miguel G
MG
Flynn
Helen R
HR
Skehel
J Mark
JM
West
Stephen C
SC
D004247:DNA / Q000737:chemistry / Q000378:metabolism; D004260:DNA Repair; D006367:HeLa Cells; D045565:Holliday Junction Resolvases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D006801:Humans; D011995:Recombination, Genetic; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity
D029701:Saccharomyces cerevisiae Proteins; D004247:DNA; C533748:GEN1 protein, human; D045565:Holliday Junction Resolvases; C533749:Yen1 protein, S cerevisiae