Adenosine Triphosphatase Associated with Adenosine Triphosphate-Dependent Deoxyribonuclease

Abstract

An ATPase activity that is completely dependent on DNA is associated with the ATP-dependent DNase (recB-recC enzyme) purified from Escherichia coli.There is a strong correlation between the ATPase and the DNase activities under various assay conditions. With E. coli DNA as substrate, 8-9 molecules of ATP are hydrolyzed to ADP and inorganic phosphate for every phosphodiester bond hydrolyzed by the DNase. The possible functional relationship of the ATPase and DNase activities is discussed.We reported the partial purification and characterization of an ATP-dependent DNase from Escherichia coli (1). Since this enzyme activity is absent in extracts of certain types of recombination-deficient mutant strains (recB and recC) of E. coli (1-4), it is reasonable to speculate that this enzyme is responsible for a certain step in the recombination process.The ATP-dependent DNase is an enzyme of large molecular weight (300,000-350,000), and appears to be determined by at least two genes (recB and recC), suggesting that it is composed of subunits. As a first step in elucidating the gene-product (protein) relationship and the molecular mechanism of the enzyme action, we have searched for an additional enzyme activity that may be associated with this enzyme.We now report the presence of a DNA-dependent ATPase activity associated with this DNase, and some of the basic characteristics of the ATPase. A preliminary report of this study has appeared.$ MATERIALS AND METHODSMedium for Bacterial Growth. G medium contains the following in 1 liter: 3 g beef extract, 6 g yeast extract, 10 g peptone, 0.5 g K2HPO4, 0.1 g MgSO4, 5 g NaCl, and 2 g glucose (autoclaved separately). Antifoam A (0.1 ml) was added to the fresh medium after inoculation with an overnight culture. For the standard assay, E. coli DNA was used as a substrate for the DNase. The reaction mixture was incubated for 20 min at 370C unless otherwise specified, and the reaction was terminated by the addition of 0.1 ml of a carrier solution (5 mg/ml of DNA and 10 mg/ml of bovine serum albumin) and 0.5 ml of cold 3.5% perchloric acid.After 5 min at 0C followed by centrifugation (2000 X g for 5 min), all the supernatant fluid was transferred to a planchet and, after addition of one drop of 5 N KOH, the sample was evaporated to dryness. The radioactivity was measured by a gas-flow counter model 4342 detectable phosphatase activity in the purified enzyme preparation, 32P-labeled DNA can be used for the assay of ATPase activity instead of unlabeled DNA). After 20 min of incubation at 370C, the reaction was terminated with 4 ml of 1.25 N perchloric acid containing 1 Mmol/ml of monosodium phosphate, followed by 4 ml of isobutanol-benzene (1:1) and 0.5 ml of 10% ammonium molybdate solution. After the whole solution was mixed with a vortex mixer, 1 ml of the 15

The recBC Deoxyribonuclease of Escherichia coli: Isolation and Characterization of the Subunit Proteins and Reconstitution of the Enzyme

“…The enzyme used here was prepared by the method of Nobrega et al (9), with omission of DEAE-cellulose chromatography.…”

“…The recBC DNase and ATPase were assayed as described (9,10) and heat stability (half-life 30 min at 47.5°) as the original enzyme (data not shown). These results suggested that the DNase molecules were dissociated into two subunits by the NaCl treatment and that active enzvme molecules were reconstituted when these subunits were recombined.…”

A third DNA‐dependent ATPase from Bacillus cereus free of ATP‐dependent DNase activity

“…The inactivation of ATP-dependent DNase during DEAE-cellulose chromatography was observed by several authors [10,15,17,20,31,32]. The extent of inactivation differed, depending on the chromatographic resolutions used.…”

“…Enzymes which degrade ATP to ADP and Pi using DNA as cofactur have been detected in both prokaryotic El- 31 and eukaryotic cells [4,5]. Several publications deal witb enzymatic reactions implied in replication and recombination of the genetic material in which ~NA-dependent hydrolysis of ATP occurs [1,2,3,6].…”

Carcinogen‐Induced DNA Repair in Nucleotide‐Permeable Escherichia coli Cells

“…This genetic exchange of preexisting intact DNA scqucnces is accompanied by DNA repair synthesis at the insertion sites [50,51]. It has been shown that the recombinational genes involved, recB and recC, specify and ATP-dependent nuclease [52] (exonuclease V) which degrades single-stranded and duplex DNA in the 3' + 5' and 5' --t 3' directions. The ATP requirement of the (Ac),ONFln-induced and 7-bromomethylbenz[u]anthracene-induced DNA synthesis could indicate the involvement of this enzyme in the DNA exchange process, e.g., in expanding post-replication gaps before donor strands are inserted.…”