Isolation, characterization, and activation of the magnesium-dependent endodeoxyribonuclease from Bacillus subtilis

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Abstract
A major endo-DNase was isolated from a mutant of the transformable B. subtilis 168. The Mg-dependent endonuclease was purified .apprx. 750-fold to electrophoretic homogeneity. The enzyme had a MW of .apprx. 31,000 as determined by gel filtration and polyacrylamide gel electrophoresis. The protein appears to be composed of 2 subunits. The nuclease was dependent on Mg2+ or Mn2+ for hydrolytic activity. The purified nuclease degraded DNA from several Bacillus spp.; Escherichia coli DNA; alkylated, depurinated and thymine-dimer containing B. subtilis DNA; and hydroxymethyluracil-containing phage DNA. The enzyme also hydrolyzed single-stranded DNA, although native DNA was the preferred substrate. The nuclease was unable to degrade rRNA. The cleavage products of the DNA hydrolysis have 5''-phosphate and 3''-hydroxyl ends. The enzyme could be activated in crude extracts by heat treatment or treatment with guanidine hydrochloride. The nuclease activity was inhibited by phosphate and by high concentrations of NaCl. A possible function for this endonuclease in bacterial transformation is discussed.