Reactions of mitochondrial cruciform cutting endonuclease 1 (CCE1) of yeast Saccharomyces cerevisiae with branched DNAs in vitro.
Cruciform-cutting endonuclease 1 (CCE1) is an X-solvase from yeast Saccharomyces cerevisiae [Kleff, S., Kemper, B. & Sternglanz, R. (1992) EMBO J. 11, 699-704]. We report here the purification of the cloned enzyme CCE1 to near homogeneity from over-expressing Escherichia coli cells. The purified protein has a globular shape and an apparent molecular mass of 38 kDa. CCE1 reacts specifically with branched DNAs, preferably with four-armed cruciforms. The enzyme linearizes native supercoiled DNA by cutting at the base of cruciform structures as they occur in derivatives of phage M13. Supercoiling was not required for cleavage per se and a relaxed circular DNA hybrid with a stable cruciform was linearized with the same relative cleavage efficiency. Fully synthetic cruciforms (four-armed X-junctions) were also good substrates for CCE1, provided a symmetric 6-bp sequence (in our case an EcoRI restriction site) was maintained at the junction. Consequently, a synthetic cruciform made from fully randomized oligonucleotide sequences was not a substrate for CCE1. In general, cleavage sites were found clustered in a characteristic pattern in each arm of a cruciform structure. A synthetic three-armed Y-junction was also cleaved by CCE1, but with a lower efficiency than the related four-armed construct. CCE1 resolves efficiently branched synthetic DNAs in vitro. The function is consistent with the idea that CCE1 is responsible for a timely reversal of branched recombination intermediates preceding petite formation in mitochondrial DNA.
Kupfer
C
C
Institut für Genetik, Köln, Germany.
Kemper
B
B
D017122:Bacteriophage T4 / Q000201:enzymology; D001483:Base Sequence; D004279:DNA, Viral / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D004706:Endodeoxyribonucleases / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism*; D045565:Holliday Junction Resolvases; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009690:Nucleic Acid Conformation; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins; D013379:Substrate Specificity
D004279:DNA, Viral; D029701:Saccharomyces cerevisiae Proteins; D004706:Endodeoxyribonucleases; C473486:CCE1 protein, S cerevisiae; D045565:Holliday Junction Resolvases; C035571:endodeoxyribonuclease VII