Susceptibility of Dinucleotides Bearing Either 3'- or 5'-Monophosphate to Micrococcal Nuclease

Abstract

Abstract A new simplified procedure for preparation of micrococcal nuclease (Staphylococcus aureus strain foggi) in good yield is described. The method consists of (a) chromatography on carboxymethyl cellulose, (b) fractionation with ammonium sulfate, (c) gel filtration on Bio-Gel P-100, and (d) chromatography on phosphocellulose. The product obtained contains no more than 2 x 10-6 unit of monophosphatase per unit of nuclease. Specific activity of nuclease across the chromatographic peak of the last step is constant. In agreement with other laboratories, the purified nuclease shows one major and one minor band on acrylamide gel electrophoresis. When this preparation of micrococcal nuclease was used in concentrations approaching saturation, deoxyribodinucleotides, which up until now have been considered resistant, were all digested. Of 12 dinucleotides of the type d-NpNp, the most susceptible was d-TpTp, the most resistant d-TpGp. The extremes in susceptibility are of the order of 100-fold and depend primarily on the base in the β position. Dinucleoside monophosphates are also digestible but are more resistant than their 3'-monophosphoryl analogues. The 5'-dinucleotides are the most resistant of all. The magnitude of inhibition by 5'- and of enhancement by 3'-phosphoryl groups is no greater than the effect exerted by the base.