Holliday junction resolvase in Schizosaccharomyces pombe has identical endonuclease activity to the CCE1 homologue YDC2.
A novel Holliday junction resolving activity has been identified in fractionated cell extracts of the fission yeast Schizosaccharomyces pombe . The enzyme catalyses endonucleolytic cleavage of Holliday junction-containing chi DNA and synthetic four-way DNA 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, linear duplex DNA or duplex DNA containing six mismatched nucleotides in the centre. The major cleavage sites map as single nicks in the vicinity of the crossover point, 3' of a thymidine residue. These data indicate that the activity has a strong DNA structure selectivity as well as a limited sequence preference; features similar to the Holliday junction resolving enzymes RuvC of Escherichia coli and the mitochondrial CCE1 (cruciform-cuttingenzyme 1) of Saccharomyces cerevisiae. A putative homologue of CCE1 in S.pombe (YDC2_SCHPO) has been identified through a search of the sequence database. The open reading frame of this gene has been cloned and the encoded protein, YDC2, expressed in E.coli . The purified recombinant YDC2 exhibits Holliday junction resolvase activity and is, therefore, a functional S.pombe homologue of CCE1. The resolvase YDC2 shows the same substrate specificity and produces identical cleavage sites as the activity obtained from S. pombe cells. Both YDC2 and the cellular activity cleave Holliday junctions in both orientations to give nicks that can be ligated in vitro. The partially purified Holliday junction resolving enzyme in fission yeast is biochemically indistinguishable from recombinant YDC2 and appears to be the same protein.
Oram
M
M
Department of Biochemistry and Molecular Biology, University College London, London WC1E 6BT, UK.
Keeley
A
A
Tsaneva
I
I
D000595:Amino Acid Sequence; D001483:Base Sequence; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D019281:Dimerization; D004706:Endodeoxyribonucleases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004720:Endonucleases / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D045565:Holliday Junction Resolvases; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000378:metabolism; D011995:Recombination, Genetic; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D012568:Schizosaccharomyces / Q000201:enzymology*; D029702:Schizosaccharomyces pombe Proteins; D016415:Sequence Alignment
D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D029702:Schizosaccharomyces pombe Proteins; D004247:DNA; D004706:Endodeoxyribonucleases; D004720:Endonucleases; C473486:CCE1 protein, S cerevisiae; D045565:Holliday Junction Resolvases; C108766:cce1 protein, S pombe