Purification of a eukaryotic site-specific endonuclease, Endo.Sce I, from Saccharomyces cerevisiae and effectors on its specificity and activity.
A site-specific endonuclease (Endo.Sce I) which caused double-strand scission of DNA was highly purified from a eukaryote, Saccharomyces cerevisiae IAM4274. The molecular weight of the active form of Endo.Sce I was estimated to be 120,000 and 110,000 by sedimentation analysis on a glycerol density gradient and gel filtration on Ultrogel AcA34, respectively. Analysis of the fractions from the last column chromatography by polyacrylamide gel-electrophoresis in the presence of sodium dodecyl sulfate and by an assay of the endonucleolytic activities suggested that Endo.Sce I consists of two non-identical subunits with molecular weights of 75,000 and 50,000. Unlike restriction endonucleases, Endo.Sce I was active on chromosomal DNA of the cells which produced Endo.Sce I. Single-stranded DNA was not cleaved by Endo.Sce I, but inhibited the endonucleolytic activity of the enzyme on double-stranded DNA. The endonucleolytic activity of Endo.Sce I required the magnesium ions (Mg2+) as a sole cofactor; Mg2+ could not be replaced by Ca2+ or Zn2+. When Mg2+ was replaced by manganese ions (Mn2+), extensively purified Endo.Sce I cleaved double-stranded DNA at many other sites in addition to the sites at which DNA was cleaved in the presence of Mg2+. Experiments indicated that this is not the activation of contaminating endonuclease in the preparation of Endo.Sce I, but the result of relaxation in the site-specificity of cleavage.
D001483:Base Sequence; D002413:Cations, Divalent; D004262:DNA Restriction Enzymes / Q000302:isolation & purification* / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific; D004706:Endodeoxyribonucleases; D007700:Kinetics; D046911:Macromolecular Substances; D008274:Magnesium / Q000494:pharmacology; D008970:Molecular Weight; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins; D013379:Substrate Specificity
D002413:Cations, Divalent; D046911:Macromolecular Substances; D029701:Saccharomyces cerevisiae Proteins; D004706:Endodeoxyribonucleases; D004262:DNA Restriction Enzymes; C037725:SCEI protein, S cerevisiae; D015252:Deoxyribonucleases, Type II Site-Specific; D008274:Magnesium