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 Bacillus 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 singlestranded DNA (separated strands of B. subtilis DNA and linear phage fd DNA) is not attacked, neither are there any circular forms (supercoiled or nicked simian virus 40 and circular single-stranded fd DNAs). The doublestranded DNA can be completely hydrolysed, 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 processively liberating mononucleotides from both strands of the native linear DNA. ATP (indispensable for the DNase reaction) is also hydrolysed by the enzyme, to ADP and inorganic orthophosphate (Pi) in the presence of DNA. The apparent K, 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.

Characterization of Bacillus subtilis recombinational pathways

“…The addAS and addB72 mutant strains have impairments in different subunits of the AddBCD enzyme (3,11,12,19). When the addAS mutation was introduced into the addB72 genetic background, we observed that the double mutant strains were as deficient as the addAS parent strain ( Table 2).…”

“…The most economical assumption is that the addA and addB mutations define an epistatic group. In the absence of the AddABC enzyme (addA5 or addA5 addB72 mutant strains), there was still a high residual level of recombination in B. subtilis (3,11,13) (Table 2). We have ruled out the possibility that the mutant enzymes retained their recombination activity, because many addA5 double mutant strains (Table 2) were deficient in recombination.…”

“…Genetic studies of these mutants have defined 12 different genes (25). The biochemical activities of some of those genes, e.g., recE (24) and addABC (11,12,27,29), have been defined. The recF and recM loci have only been preliminarily characterized (1)(2)(3)17).…”

“…Functions associated with the AddABC enzyme (also designated Exo V) include a processive ATP-dependent double-strand exonuclease, a single-strand exonuclease, a DNA-dependent ATPase, and an ATP-dependent doublestrand-unwinding enzyme (11,27,29,32). The AddBCD protein is the B. subtilis counterpart of the E. coli RecBCD enzyme (19,32).…”

Characterization of recombination-deficient mutants of Bacillus subtilis

“…A processive double-strand exonuclease, a single-strand exonuclease, a DNA-dependent ATPase, and an ATP-dependent double-strand unwinding function were associated with the purified B. subtilis ExoV analog (12,32,38) as they were with E. coli ExoV (15).…”