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 recombinat ion. We describe the identification and initial biochemical characteri sation of a new Holliday junction resolvase from Schizosaccharomyces p ombe. 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 polarit y. All cuts occurred 3' of thymine nucleotides with a possible prefere nce 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 ide ntified in the database (SpCCE1). The gene was cloned by PCR, overexpr essed in Escherichia coli and its product purified as a His-tagged fus ion protein. Purified SpCCE1 binds to X-junctions in a structure-speci fic manner and resolves them to nicked linear duplex products that are repairable by DNA ligase. SPCCE1 cuts X-junctions in precisely the sa me 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 i ts ability to complement a resolvase-deficient strain of E. coli. (C) 1997 Academic Press Limited.