The nucleases of yeast: II. Purification, properties and specificity of an endonuclease from yeast

Abstract

A nuclease has been purified approx. 30-fold from the supernatant fraction of a hybrid yeast ( Saccharomyces fragilis × Saccharomyces dobzhanskii) by salt fractionation, chromatography on Sephadex G-200, and DEAE-cellulose chromatography. The optimum pH is 7.6 and Mg 2+ is required for the full activity. The action of this nuclease on polyribonucleotides is exclusively endonucleolytic. The major products upon extensive digestion of homopolymers are di- and trinucleotides having 5′-phosphomonoester end groups. The formation of mononucleotide is slight. Polyadenylic acid and polyuridylic acid are hydbrolyzed first to a family of small oligonucleotides having 5′-phosphomonoester end groups. The distribution of these oligonucleotides is not completely random and depends on the conformation of the substrate. Hydrolysis of shorter chains is much slower than longer chains. This endonuclease has no apparent specificity for a particular base residue in polynucleotides. This mode of hydrolysis coupled with its high stability make this enzyme a very useful reagent for the preparation of oligonucleotides with 5′-phosphate ends from either synthetic polynucleotides or nature RNA. This enzyme appears to be specific for polynucleotides having a random coil conformation. Double- and triple-stranded helical conformations are less susceptible to attack. Polyadenylic acid, polyuridylic acid and polyinosinic acid are hydrolyzed much faster than polycytidylic acid, transfer RNA or ribosomal RNA. The enzyme preparation hydrolyzes denatured DNA at approximately the same rate as yeast ribosomal RNA. Highly polymerized native Escherichia coli DNA is almost inactive as substrate.