Site-Specific Endo-Deoxyribonucleases in Eukaryotes: Endonucleases of YeastsSaccharomyces and Pichia1

A class of endo-deoxyribonucleases was found in cell-free extracts from yeasts; Saccharomyces cerevisiae, S. uvarum and Pichia membranaefaciens. These endonucleases cleaved double-stranded DNA so that the treated DNA exhibited a set of discrete bands on an agarose gel-electrophoregram. The proffles obtained with a certain DNA were unique to each endonuclease, suggesting that these yeast endo nucleases cleave DNA at well-defined sites specific to each endonuclease. We partially purified one of the yeast endonucleases from S. cerevisiae IAM4274, endonuclease (Endo.) Sce I. Endo. Sce I, whose molecular weight was determined to be approximately 90,000 daltons by gel-filtration, is active on either linear DNA of coli phage λ and Bacillus phages or superhelical closed circular DNA of pBR322. The phage DNA treated with Endo. Sce I exhibited discrete bands on an agarose gel-electrophoregram whether the product was denatured with 0.1 N NaOH or not. These observations suggest that Endo.Sce I introduced a limited number of double-strand scissions at unique sites on the DNA. Double-stranded DNA fragments obtained by the cleavage with Endo.Sce I were ligated with T4-DNA ligase. This observation indicates that the fragments have flush or cohesive ends bearing free 3′-hydroxyl and 5′-phosphoryl groups. This result also indicates that double-strand scissions are not introduced by accumulation of single-strand scissions or gaps. Endo. Sce I seems to introduce a single-strand scission in one of the strands at a unique site followed by the introduction of a second scission nearby in the other strand of closed circular double-stranded DNA of pBR322 to produce a linear form with unit length. The cleavage site on pBR322 DNA with Endo. Sce I was located in the vicinity of the cleavage site for restriction endonuclease HindIII. Magnesium ion was required for the nucleolytic activity and could not be replaced by calcium ion. The nucleolytic activity was not stimulated by NaCl or KCI but was inhibited above 100 m We did not detect any effect of ATP, spermidine, novobiocin or nalidixic acid on the nucleolytic activity of Endo. Sce I.