HOLLIDAY JUNCTION RESOLVASE IN SCHIZOSACCHAROMYCES-POMBE HAS IDENTICAL ENDONUCLEASE ACTIVITY TO THE CCE1 HOMOLOG YDC2

A novel Holliday junction resolving activity has been identified in fr actionated cell extracts of the fission yeast Schizosaccharomyces pomb e. The enzyme catalyses endonucleolytic cleavage of Holliday junction- containing chi DNA and synthetic four-way DMA junctions, The activity cuts with high specificity a synthetic four-way junction containing a 12 bp core of homologous sequences but has no activity on another four -way junction with a fixed crossover point), a three-way junction, lin ear duplex DNA or duplex DNA containing six mismatched nucleotides in the centre, The major cleavage sites map as single nicks in the vicini ty of the crossover point, 3' of a thymidine residue, These data indic ate that-the activity has a strong DNA structure selectivity as well a s a limited sequence preference; features similar to the Holliday junc tion resolving enzymes RuvC of Escherichia coli and the mitochondrial CCE1 (cruciform-cutting enzyme 1) of Saccharomyces cerevisiae, A putat ive homologue am CCE1 in S.pombe (YDC2 SCHPO) has been identified thro ugh a search of the sequence database, The open reading frame of this gene has been cloned and the encoded protein, YDC2, expressed in E.col i. The purified recombinant YDC2 exhibits Holliday junction resolvase activity and is, therefore, a functional S.pombe homologue of CCE1, Th e resolvase YDC2 shows the same substrate specificity and produces ide ntical cleavage sites as the activity obtained from S.pombe cells, Bot h YDC2 and the cellular activity cleave Holliday junctions in both ori entations to give nicks that can be ligated in vitro, The partially pu rified Holliday junction resolving enzyme in fission yeast is biochemi cally indistinguishable from recombinant YDC2 and appears to be the sa me protein.