Cruciform-cutting endonuclease 1 (CCEI ) is an X-solvase from yeast Saccharomyces cerevisiae [Kleff, S., Kemper, B. & Sternglanz, R. (1992) EMBO J. 11, 699-7041, We report here the purification of the cloned enzyme CCEl to near homogeneity from over-expressing Escherichia coli cells. The purified protein has a globular shape and an apparent molecular mass of 38 kDa. CCEl 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 CCEl, 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 CCEl, but with a lower efficiency than the related four-armed construct. CCEI resolves efficiently branched synthetic DNAs in vitro. The function is consistent with the idea that CCEl is responsible for a timely reversal of branched recombination intermediates preceding petite formation in mitochondrial DNA.

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