Triphosphate-dependent Deoxyribonuclease Research Articles

Purification and properties of adenosine 5'-triphosphate-dependent deoxyribonuclease from Thermus thermophilus HB8.

An ATP-dependent DNase has been purified from Thermus thermophilus HB8 by a procedure involving streptomycin precipitation, DEAE-cellulose chromatography, Sephadex G-200 gel filtration and heparin-agarose affinity chromatography. ATP-dependent DNase activity was separated into two distinct peaks, Peak A and Peak B, by heparin-agarose affinity chromatography. Each peak fraction was further purified by ATP-agarose affinity chromatography. Peak A and Peak B were eluted from an ATP-agarose column at 0.14 M and 0.28 M KCl, respectively, each as a single peak. Both enzyme activities require ATP and Mg2+ for the degradation of double- and single-stranded DNAs, and degrade denatured DNA about 1.5 times faster than native DNA. The two peaks are optimally active at 69 degrees C and have similar optimal pH ranges from 8.2 to 9.2. The two purified peaks were unstable on storage at -20 degrees C, but were remarkably stabilized by addition of 0.4 mg/ml bovine serum albumin. Ammonium sulfate strongly inhibits the activities of both peaks. The molecular weights of Peak A and Peak B are about 170,000 as estimated by glycerol gradient sedimentation. The average chain lengths of denatured DNA produced by Peak A and Peak B were 4.2 and 3.6, respectively, and the products were terminated by 5'-phosphoryl and 3'-hydroxyl groups. The limit-digested products of denatured DNA produced by Peak B consist of mono-, di-, tri-, tetra-, and pentanucleotides along with some larger fragments. The mode of action of both activities is processive and Peak A does not attack double-stranded circular DNA.

The infectivity of adenovirus 5 DNA-protein complex

Adenovirus 5 DNA-protein complexes were prepared by treating virions with 4 M guanidinium chloride and resolving the faster sedimenting viral DNA from released capsid protein. These complexes were characterized by both gel electrophoresis and electron microscopy. After dialysis into saline buffer, approximately one-half of the viral DNA-protein complex was aggregated by association at its termini into conformations other than linear monomer length duplex molecules. However, the monomer length linear molecules in the viral DNA-protein complex sample also have protein attached to both of their termini: These linear molecules are resistant to digestion by a pressessive nuclease, adenosine triphosphate-dependent deoxyribonuclease, that requires a free terminus for activity, while Pronase-treated adenovirus DNA is susceptible to digestion, and the restriction endonuclease cleavage fragments from both ends of the viral DNA-protein complex migrate at an anomalous rate during electrophoresis in an agarose gel. As the infectivity of Ad5 DNA is exceptionally low, the infectivity of the DNA-protein complex was tested using the calcium technique for transfection. The efficiency of transfection with the Ad5 DNA-protein complex is about 100-fold higher than that of Pronase-treated Ad5 DNA.

Effect of adenosine 5'-triphosphate-dependent deoxyribonuclease deficiency on properties and transformation of Haemophilus influenzae strains.

A transformation-deficient strain of Haemophilus influenzae, lacking adenosine 5'-triphosphate-dependent deoxyribonuclease activity, was isolated by selection for sensitivity to mitomycin. The mutant, designated JK57, possibily showed a moderate sensitivity to ultraviolet (UV) irradiation and treatment with methyl methane sulfonate. Contrary to the wild type, the mutant degraded chromosomal deoxyribonucleic acid (DNA) to some extent. However, after UV irradiation to the mutant degraded considerably less DNA than the wild type and the TD24 mutant of H. influenzae, the latter being equivalent to a recA mutant of Escherichia coli. A TD2457 double mutant, constructed by transferring the TD24 mutation into the JK57 strain, was as sensitive to deleterious agents and as deficient in transformation as the TD24 single mutant; in the double mutant, however, after UV irradiation chromosomal DNA was degraded to the same extent as in the JK57 mutant. The number of transformants per unit of radioactive donor DNA taken up by JK57 recipient cells was approximately 10-fold smaller than in the wild type. Presynaptically, the fate of donor DNA in the adenosine 5'-triphosphate-dependent deoxyribonuclease-deficient mutants was not different from that in the wild type. In contrast to TD24 and the TD2457 double mutant, in the JK57 mutant, recombinant-type activities (molecules carrying both the donor and recipient markers) were formed almost as well as in the wild type. After integration into the JK57 recipient genome, the rate of replication of the donor marker was equal to that of the recipient marker during a number of generations, which suggests that the donor DNA is normally integrated into the JK57 chromosome. It is suggested that transformed JK57 cells pass with a high frequency into a type of cells that can replicate their chromosomes many times but have lost the ability to form visible colonies after plating.

Membrane location of a deoxyribonuclease implicated in the genetic transformation of Diplococcus pneumoniae

The cellular localization of enzymes in Diplococcus pneumoniae was examined by fractionation of spheroplasts. A deoxyribonuclease implicated in the entry of deoxyribonucleic acid (DNA) into the cell during genetic transformation was located in the cell membrane. This enzyme, the major endonuclease of the cell (endonuclease I), which is necessary for the conversion of donor DNA to single strands inside the cell and oligonucleotides outside, thus could act at the cell surface. Another enzyme, the cell wall lysin (autolysin), was also found in the membrane fraction. Other enzymes, including amylomaltase, two exonucleases, and adenosine triphosphate-dependent deoxyribonuclease, and a restriction type endonuclease, were located in the cytosol within the cell. None of the enzymes examined were predominantly periplasmic in location. Spheroplasts were obtained spontaneously on incubation of pneumococcal cells in concentrated sugar solutions. The autolytic enzyme appears to be involved in this process. Cells that were physiologically competent to take up DNA formed osmotically sensitive spheroplasts two to three times faster than cells that were not in the competent state. Although some genetically incompetent mutants also formed spheroplasts more slowly, other such mutants formed them at the faster rate.

Isolation and characterization of mutants of Haemophilus influenzae deficient in an adenosine 5'-triphosphate-dependent deoxyribonuclease activity

By a direct assay approach, mutants of Haemophilus influenzae Rd that are deficient in adenosine 5'-triphosphate-dependent deoxyribonuclease activity (add-) were isolated and characterized. A large proportion (50 to 90%) of the cells in cultures of these mutants failed to produce visible colonies when plated. An extensive analysis of the recombination proficiency of these strains revealed that the transformation frequency (transformants per competent cell) in the mutants was similar to that found in the wild type, but that the transformation efficiency (transformants per microgram of irreversibly bound deoxyribonucleic acid [DNA]) was reduced approximately fourfold. Sensitivities of the mutants to gamma rays, ultraviolet radiation, and methyl methane sulfonate were only slightly greater than wild-type levels. The rate of degradation of host DNA after ultraviolet irradiation was significantly reduced in the mutants. It is suggested that the adenosine 5'-triphosphate-dependent deoxyribonuclease in H. influenzae plays a nonessential role in DNA recombination and repair.

Complexes between Deoxyribonucleic Acid and the Adenosine Triphosphate-Dependent Deoxyribonuclease from Mycobacterium smegmatis

Complexes between Deoxyribonucleic Acid and the Adenosine Triphosphate-Dependent Deoxyribonuclease from <i>Mycobacterium smegmatis</i>

Increased Deoxyribonucleic Acid Polymerase and Adenosine Triphosphate-Dependent Deoxyribonuclease Activity in Mycobacterium smegmatis after Treatment with Alkylating Agents

Increased Deoxyribonucleic Acid Polymerase and Adenosine Triphosphate-Dependent Deoxyribonuclease Activity in <i>Mycobacterium smegmatis</i> after Treatment with Alkylating Agents

Fate of transforming deoxyribonucleic acid after uptake by competent Bacillus subtilis: phenotypic characterization of radiation-sensitive recombination-deficient mutants.

A collection of 16 isogenic recombination-deficient strains of Bacillus subtilis isolated on the basis of sensitivity to methyl methane sulfonate (MMS) or mitomycin C (MC) were characterized phenotypically. All were found to be somewhat sensitive to ultraviolet irradiation, MC, and MMS. The mutants were all blocked in "late" steps in the transformation process and were provisionally grouped into four categories on the basis of the various properties examined. Class I mutants were deficient in transformation and heterologous transduction with phage PBS1 but were transducible with homologous donors at nearly the wild-type frequency. They were blocked in donor-recipient complex (DRC) formation but formed essentially normal amounts of double-strand fragments (DSF) and single-strand fragments (SSF). The class IIa strain was deficient in transformation and PBS1 transduction, and formed DRC which was normal by all available physical and biological criteria. Class IIb mutants were deficient in transformation and PBS1 transduction, and failed to form DRC. They did produce DSF and SSF. Class III mutants were deficient in transformation, were normal in PBS1 transduction, and formed DRC which was physically indistinguishable from that of the Rec(+) parent although with slightly lowered donor-type transforming activity. Class IV strains were deficient in PBS1 transduction but were transformed at nearly the wild-type efficiency. None of the mutant strains was deficient in the adenosine triphosphate-dependent deoxyribonuclease.

Adenosine triphosphate-dependent deoxyribonuclease from Diplococcus pneumoniae: fate of transforming deoxyribonucleic acid in a strain deficient in the enzymatic activity.

The adenosine triphosphate-dependent deoxyribonuclease is required for wild-type levels of deoxyribonucleic acid (DNA) repair and genetic recombination. In an attempt to determine the physiological function of this enzyme in pneumococcal transformation, the fate of transforming DNA was followed in a wild-type strain and in a strain lacking the enzymatic activity. The qualitative and quantitative findings were closely comparable in the two strains through the step of physical association of a single strand of donor DNA with the recipient chromosome. These results are interpreted to mean that the enzyme may be involved in the subsequent hypothetical removal of excess polynucleotide sequences during conversion of the presumed hydrogen-bonded intermediate into a covalently linked recombinant structure.

Effects of Reaction Conditions on the Deoxyribonuclease and Adenosine Triphosphatase Activities of an Adenosine Triphosphate-Dependent Deoxyribonuclease from Mycobacterium smegmatis

Conference Article| January 01 1973 Effects of Reaction Conditions on the Deoxyribonuclease and Adenosine Triphosphatase Activities of an Adenosine Triphosphate-Dependent Deoxyribonuclease from Mycobacterium smegmatis F. G. WINDER; F. G. WINDER 1Department of Biochemistry, Trinity College, Dublin 2, Irish Republic Search for other works by this author on: This Site PubMed Google Scholar P. A. SASTRY; P. A. SASTRY 1Department of Biochemistry, Trinity College, Dublin 2, Irish Republic Search for other works by this author on: This Site PubMed Google Scholar G. R. CAMPBELL G. R. CAMPBELL 1Department of Biochemistry, Trinity College, Dublin 2, Irish Republic Search for other works by this author on: This Site PubMed Google Scholar Biochem Soc Trans (1973) 1 (1): 270–271. https://doi.org/10.1042/bst0010270 Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Twitter LinkedIn Cite Icon Cite Get Permissions Citation F. G. WINDER, P. A. SASTRY, G. R. CAMPBELL; Effects of Reaction Conditions on the Deoxyribonuclease and Adenosine Triphosphatase Activities of an Adenosine Triphosphate-Dependent Deoxyribonuclease from Mycobacterium smegmatis. Biochem Soc Trans 1 January 1973; 1 (1): 270–271. doi: https://doi.org/10.1042/bst0010270 Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search nav search search input Search input auto suggest search filter All ContentAll JournalsBiochemical Society Transactions Search Advanced Search This content is only available as a PDF. © 1973 Biochemical Society1973 Article PDF first page preview Close Modal You do not currently have access to this content.

Adenosine triphosphatase associated with adenosine triphosphate-dependent deoxyribonuclease (recB-recC enzyme-E. coli-ATP to phosphodiester hydrolysis ratio-DNA-dependent ATPase activity).

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.

Partial purification and properties of a nucleoside triphosphate-dependent deoxyribonuclease from Mycobacterium smegmatis

An ATP-dependent deoxyribonuclease was partially purified from iron-limited Mycobacterium smegmatis. The enzyme showed a strong preference for native DNA, and was completely dependent on the presence of a riboside, or deoxyriboside, triphosphate and Mg 2+. Maximal activity was reached with about 1 mg of sodium DNA per ml, 4 mM ATP and 30 mM Mg 2+. In the presence of DNA the enzyme hydrolyzed ATP to ADP and orthophosphate, one mole of ATP being hydrolyzed per mole of deoxyribonucleotide solubilized. The DNA was attacked from the ends of strands, and the evidence favoured attack on both strands. The products released from DNA were 4–13 % mononucleotides, 14–26 % dinucleotides, 29–45 % trinucleotides, 15–23 % tetranucleotides and 5–29 % of longer oligonucleotides, as fractionated on DEAE-Sephadex. Fractionation on Sephadex G-25, on DEAE-paper, and by two-dimensional paper chromatography gave results in keeping with this. The properties of the enzyme are discussed in the light of a possible role in recombination.

The Isolation and Characterization from Escherichia coli of an Adenosine Triphosphate-dependent Deoxyribonuclease Directed by rec B, C Genes

Abstract Extracts of wild type Escherichia coli contain a DNase which catalyzes the release of acid-soluble fragments from native DNA; this activity cannot be detected in rec B and C mutants. The properties of this enzyme have been studied with a fraction purified 200- to 600-fold. Only in the presence of ATP does the purified enzyme act on native DNA; with denatured DNA this ATP requirement is not absolute. The purified fraction requires Mg++ and is active over a broad pH range with an optimum of about 9.2. The exonuclease action of the purified enzyme fraction is due to two exonuclease activities. (a) An exonuclease, which we have called exonuclease V, acts on double-stranded DNA only in the presence of an activating nucleoside triphosphate, ATP and dATP being the most efficient. During DNA degradation, ATP is cleaved to ADP and Pi. The products formed during the degradation of native DNA are composed of 3'-hydroxyl- and 5'-phosphate-ended oligonucleotides, the average size of which decreases as the reaction proceeds. Exonuclease V attacks DNA from ends generated by double-stranded breakage and does not attack circular double-stranded DNA molecules with or without single-stranded breaks. Also associated with exonuclease V is an ATP-dependent cleavage of single-stranded DNA. (b) A 3'-exonuclease, acts on single-stranded DNA in the absence of ATP, and whose properties are distinguishable from those of exonuclease I, is responsible for the other exonuclease activity. The possible role of exonuclease V in DNA recombination is discussed.

An ATP-dependent deoxyribonuclease from Diplococcus pneumoniae: I. Partial purification and some biochemical properties

1. 1. A 5-step procedure is described for the partial purification of a nucleoside triphosphate-dependent deoxyribonuclease from Diplococcus pneumoniae. An overall purification of more than 500-fold was achieved. 2. 2. The enzyme was found to be active under alkaline conditions (optimum pH from 9.25 through, at least, 9.8) and to require a metal ion and a nucleoside triphosphate for its activity on native DNA. Mg 2+ and Mn 2+ and ATP were found to be the most effective at satisfying these requirements. 3. 3. ATP is split during the course of DNA hydrolysis. 4. 4. The zone sedimentation pattern of the DNA hydrolysis products is consistent with an enzyme that attacks preexisting free ends (exonuclease), liberating oligonucleotides among its products. 5. 5. The enzyme is much more active on native DNA than on heat-denatured DNA. It attacks poly d(A-T) but exhibits no activity with rRNA.

Chain lengths of the oligodeoxyribonucleotides formed by the nucleoside triphosphate-dependent deoxyribonuclease of Mycobacterium smegmatis.

Research Article| September 01 1970 Chain lengths of the oligodeoxyribonucleotides formed by the nucleoside triphosphate-dependent deoxyribonuclease of Mycobacterium smegmatis F G Winder; F G Winder Search for other works by this author on: This Site PubMed Google Scholar M Lavin M Lavin Search for other works by this author on: This Site PubMed Google Scholar Biochem J (1970) 119 (3): 25P–26P. https://doi.org/10.1042/bj1190025P Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Facebook Twitter LinkedIn MailTo Cite Icon Cite Get Permissions Citation F G Winder, M Lavin; Chain lengths of the oligodeoxyribonucleotides formed by the nucleoside triphosphate-dependent deoxyribonuclease of Mycobacterium smegmatis. Biochem J 1 September 1970; 119 (3): 25P–26P. doi: https://doi.org/10.1042/bj1190025P Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsBiochemical Journal Search Advanced Search This content is only available as a PDF. © 1970 The Biochemical Society1970 Article PDF first page preview Close Modal You do not currently have access to this content.

Biochemical and genetic studies of recombination proficiency in Escherichia coli. I. Enzymatic activity associated with recB+ and recC+ genes.

An adenosine triphosphate-dependent deoxyribonuclease activity has been detected in lysates of recB(+)recC(+) strains. Mutations in recB or recC lead to loss of this activity, suggesting that these two genes determine the nuclease activity. The over-all reaction in crude lysates digests native DNA to nucleoside monophosphates. Complementation between recB21 and recC22 in vivo leads to normal levels of ATP-dependent nuclease activity. No complementation in vitro has been detected. Mutations in a third recombination gene (recA) do not alter significantly the wild-type levels of this nuclease activity.