A new Holliday junction resolving enzyme from Schizosaccharomyces pombe that is homologous to CCE1 from Saccharomyces cerevisiae.
The resolution of Holliday junctions is a critical stage in recombination. We describe the identification and initial biochemical characterisation of a new Holliday junction resolvase from Schizosaccharomyces pombe. Resolvase activity was initially detected in partially purified cell-free extracts of S. pombe. Resolution of X-junction DNA occurred by the introduction of symmetrical cuts in strands of the same polarity. All cuts occurred 3' of thymine nucleotides with a possible preference for cleavage one nucleotide 3' from the point of strand crossover. During the course of these studies, a potential S. pombe homologue of the Saccharomyces cerevisiae Cruciform Cutting Endonuclease I was identified in the database (SpCCE1). The gene was cloned by PCR, overexpressed in Escherichia coli and its product purified as a His-tagged fusion protein. Purified SpCCE1 binds to X-junctions in a structure-specific manner and resolves them to nicked linear duplex products that are repairable by DNA ligase. SpCCE1 cuts X-junctions in precisely the same way as the resolvase activity from partially purified extracts of S. pombe, indicating that they are probably the same. Finally, we show that SpCCE1 can function as a Holliday junction resolvase in vivo by its ability to complement a resolvase-deficient strain of E. coli.
Whitby
M C
MC
Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU, U.K.
Dixon
J
J
D001426:Bacterial Proteins / Q000378:metabolism; D003001:Cloning, Molecular; D011088:DNA Ligases / Q000378:metabolism; D004260:DNA Repair; D004706:Endodeoxyribonucleases / Q000737:chemistry* / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology; D029968:Escherichia coli Proteins; D005816:Genetic Complementation Test; D045565:Holliday Junction Resolvases; D045522:Recombinases; D011995:Recombination, Genetic; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins; D012568:Schizosaccharomyces / Q000201:enzymology* / Q000235:genetics; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D019895:Transposases / Q000737:chemistry / Q000378:metabolism*
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D045522:Recombinases; D029701:Saccharomyces cerevisiae Proteins; C069806:ruvC protein, E coli; D019895:Transposases; D004706:Endodeoxyribonucleases; C473486:CCE1 protein, S cerevisiae; D045565:Holliday Junction Resolvases; D011088:DNA Ligases