Purification and characterization of a nutritionally controlled endodeoxyribonuclease from Streptomyces glaucescens

Abstract

Streptomyces glaucescens has a DNAase whose synthesis is under nutritional control. We have purified this enzyme to apparent homogeneity by phosphocellulose chromatography followed by heparin-agarose, Cibacron Blue F3-GA-Sepharose and Sephadex G-75 chromatography and MonoQ f.p.l.c. The enzyme had an apparent Mr of 39,600 and a pI of approx. 8.15. The Mr of the native enzyme estimated by gel chromatography was 49,000. The DNAase had a pH optimum of 7.5 and an absolute requirement for bivalent cations in the reaction buffer. It was inhibited by high salt concentrations, chelating agents or phosphate-containing compounds and was stimulated by dimethyl sulphoxide. The activity was greatly diminished unless dithiothreitol or 2-mercaptoethanol was included in the reaction mixture. Reagents such as Hg2+ or iodoacetate strongly inhibited the enzyme. The nuclease hydrolysed both double-stranded and single-stranded DNA, showing greater affinity for double-stranded DNA, and no detectable hydrolysis of RNA. The enzyme produced nicks in double-stranded DNA, generating 3′-hydroxy and 5′-phosphate termini, and degraded circular DNA.