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Purification and properties of a nuclease from Saccharomyces cerevisiae that cleaves DNA at cruciform junctions.

1987; Elsevier BV; Volume: 262; Issue: 26 Linguagem: Inglês

10.1016/s0021-9258(18)45270-2

1083-351X

Stephen C. West, Carol A. Parsons, Steven M. Picksley,

Plant Genetic and Mutation Studies

An endonuclease specific for cruciform junctions has been purified from yeast cells treated with a DNAdamaging agent.The activity was followed through five chromatographic steps by assaying for the linearization of supercoiled plasmid DNA, which extrudes cruciform structures in uitro.The sites of cleavage on pColIR215 were sequenced, and nicks were located to positions symmetrically opposed across the cruciform junction.The products of cleavage were unit length linear duplexes that contained terminal hairpin loops.In contrast to pColIR215, the cleavage patterns of pXG540 plasmid DNA were found to be complex, and cuts were found up to 4 0 bases from an (A-T)SI sequence that extrudes into a cruciform.Little or no activity could be detected on single-stranded DNA, linear duplex DNA, or nicked circular duplex DNA.The nuclease was insensitive to RNase but was inactivated by treatment with proteinase K. Mg2+ was required as cofactor and could not be replaced by Mn2+, Ca", Co2', or Cu2+.The native molecular weight of the activity was approximately 200,000 as estimated by gel filtration. Biochemical and enzymatic studies of the Escherichia coliRecA protein (for review, see Ref. 1) have led to the development of detailed molecular models for the mechanism of homologous pairing and strand exchange (2).However, the isolation and study of other enzymes, notably those required for the resolution of Holliday junctions, are required to fill important gaps in our understanding of the enzymology of genetic recombination.Although no cellular resolving protein has yet been isolated, two bacteriophage endonucleases involved in the maturation and packaging of phage DNA are known to act specifically a t branch points in DNA and cleave Holliday junctions in uitro.T4 endonuclease VI1 (the product of gene 49) and T7 endonuclease I (the product of gene 3) cut Holliday junctions and Y-junctions in DNA to produce unbranched duplexes, each with a ligatable nick (3-7).Supercoiled plasmids that contain inverted repeat sequences extrude cruciform structures in uitro (8-11).These structures are of interest because the configuration of the DNA strands at the base of the junction is analogous to that of a Holliday junction.The T4 and T7 endonucleases act specifically at the site of a cruciform and introduce nicks into regions that are symmetrically opposed across the base of the junction (4, 7).Plasmids containing cruciforms also provide a simple sub-