Isolation, Subunit Structure and Properties of the ATP‐Dependent Deoxyribonuclease of Bacillus subtilis

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Abstract
A procedure was developed for the purification of the ATP-dependent DNase [EC 3.1.4.-] of B. subtilis 168. It comprises (NH4)2 SO4 fractionation, Sephadex gel filtration, DEAE-cellulose chromatography and gel electrophoresis on a discontinuous polyacrylamide gradient. The enzyme was obtained in a homogeneous state. Its MW was 270,000 by disc electrophoresis. Dodecylsulfate-polyacrylamide gel electrophoresis showed the presence of 5 nonidentical subunits of MW 81,000, 70,000, 62,000, 52,500 and 42,500. These values give 308,000 as the MW of the native enzyme. The pH optimum of the purified enzyme is 9.6. The optimal concentrations of Mg2+ and ATP for exonuclease activity on native B. subtilis DNA were determined. ATP-requirement for hydrolysis of single-stranded DNA is less stringent. The enzyme possesses high DNA-dependent ATPase activity. The purification procedure was applied to extracts of the mutant devoid of activity for this enzyme (strain GSY 1290). A protein was isolated which is similar to the active DNase as regards electrophoretic mobility, reaction with specific antisera and size of 4 of the subunits. One subunit is missing (MW 70,000) and is replaced by smaller polypeptide (MW 56,500). The latter results suggest that the mutant is affected in the genetic locus coding for the 70,000-MW subunit.