Substrate Specificity and Adenosine Triphosphatase Activity of the ATP-Dependent Deoxyribonuclease, of Bacillus subtilis

Abstract

Studies on the specificity of the ATP-dependent DNase of B. subtilis 168, carried out with pure enzyme at the optimal conditions for its action, have shown that the substrate is double-stranded linear DNA. Linear single-stranded DNA (separated strands of B. subtilis DNA and linear phase fd DNA) is not attacked, neither are there any circular forms (supercoiled or nicked SV 40 and circular single-stranded fd DNA). The double-stranded DNA can be completely hydrolyzed, the limit products being, almost exclusively, mononucleotides. The presence of terminal phosphate residues in the substrate (either at the 3'' or the 5'' end) is not necessary for enzyme action. This DNase appears therefore to be an exonuclease progressively liberating mononucleotides from both strands of the native linear DNA. ATP (indispensable for the DNase reaction) is also hydrolyzed by the enzyme, to ADP and Pi in the presence of DNA. The apparent Km for ATP, in the ATPase reaction, is 0.15 mM. At high ATP concentrations, which inhibit the DNase activity, there is activation of the ATPase reaction. Three molecules of ATP are consumed for each DNA phosphodiester bond split, at optimal conditions for DNase activity.