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PubMed1982

Chromatin-associated deoxyribonuclease activity in liver nuclei isolated from Rana catesbeiana froglets and premetamorphic and T3-induced tadpoles.

Nuclei and chromatin isolated in the presence of calcium or magnesium from Rana catesbeiana liver tissue exhibit considerable endogenous deoxyribonuclease activity. This activity is present in liver nuclei isolated from froglets as well as in liver nuclei isolated from untreated and thyroid hormone treated premetamorphic tadpoles. Nuclei and chromatin isolated in the absence of divalent cations and in the presence of spermine exhibit no detectable expression of the endogenous deoxyribonuclease activity. The endogenous deoxyribonuclease present, but not expressed, in spermine-isolated tadpole liver nuclei or chromatin is salt extractable. Once dialyzed, the salt-extracted deoxyribonuclease is activated by calcium or magnesium. This deoxyribonuclease shows maximal enzyme activity in 15 mM calcium at pH 8.0 or in 15 mM magnesium at pH 7.4. After Ca2+ activation, deoxyribonuclease activity is maximally inhibited by amounts of spermine similar to that required to completely inhibit DNase I. Destruction of the salt-extracted deoxyribonuclease activity by treatment with proteinase K or heat suggests that it is of a proteinaceous nature. The localization and nature of this enzyme activity established that it is associated with the salt-soluble proteins affiliated with tadpole and froglet liver chromatin.

Animals↗

PubMed1982

Investigation of the actin-deoxyribonuclease I interaction using a pyrene-conjugated actin derivative.

The interaction of deoxyribonuclease I with muscle actin was studied with the aid of a pyrenyl derivative of the actin [Kouyama, T., & Mihashi, K. (1981) Eur. J. Biochem. 114, 33-38] that increases its quantum yield by an order of magnitude on polymerization. It is shown that this derivative copolymerizes with unlabeled G-actin in a random manner and will also bind to deoxyribonuclease with inhibition of enzymic activity. The derivative affords a highly sensitive means of following nucleated polymerization. Preincubation of F-actin with deoxyribonuclease at a concentration of 5% or less of that of total subunits causes inhibition of polymerization of additional G-actin onto the filaments. In red cell membranes that contain stabilized short filaments of actin such that the concentration of filament ends is large relative to monomers, complete inhibition of nucleated polymerization of G-actin is achieved by preincubation with deoxyribonuclease. The results indicate that binding of DNase occurs at the "plus" ends of the actin filaments. Competition with cytochalasin E, which is known to have a high affinity for the plus or preferentially growing ends of F-actin, can be observed. Whereas the activity of deoxyribonuclease in the 1:1 complex with G-actin is inhibited, the enzyme attached to the ends of filaments appears to be fully active. This causes a reduction in the inhibition of enzymic activity with increasing F-actin concentration, presumably by reason of a change in the partition of the enzyme between monomers and filament ends. The degree of inhibition increases with time, however, as the actin depolymerizes. Implications for measurements of actin monomer concentrations by the deoxyribonuclease assay procedure are considered.

Actins↗

PubMed1973

Penetration of a bacteriophage into Bacillus subtilis: blockage of infection by deoxyribonuclease.

Plaquing of a newly isolated phage of Bacillus subtilis, phage 41c, is only 2% efficient in agar containing 200 mug of deoxyribonuclease per ml. Timed deoxyribonuclease addition experiments showed that phage development is blocked in 90% of the cells if deoxyribonuclease is present during adsorption (zero-time samples), whereas 10 min after adsorption the enzyme has little effect (10-min samples). The fate of (32)P-deoxyribonucleic acid label of phage 41c in zero-time samples was compared to that in 10-min samples. In both, about 80% of the label remained with the phage-bacterium complex on initial centrifugation. However, four successive washings removed 90% of the (32)P from the zero-time samples but only 25% from the 10-min samples. In both samples, most of the washed-out label was of low molecular weight. When the time course of interruption of infection by blending was compared with interruption by deoxyribonuclease treatment, the two processes exhibited similar kinetics. It is postulated that both processes block injection at the same site, namely, the point of contact between phage tail and cell wall surface. Partitioning of (32)P label during protoplasting of zero-time and 10-min samples was similar to that observed during washing. For the protoplasting experiments, a quantitative method for plaquing protoplasts was developed. A single bacillus made of several cells can give rise to several protoplast plaque-forming units. Strain 41c was the only phage of seven tested to be inhibited by deoxyribonuclease. No other deoxyribonuclease-sensitive phages have been described.

Adsorption↗

PubMed1997

Application of autolysin and deoxyribonuclease profiles generated by renaturing SDS-PAGE in the comparison of selected Proteobacteria.

SDS-PAGE of cell-free extracts in gels containing bacterial murein or DNA allowed, after enzyme renaturation and staining of nonhydrolysed substrate, the detection of multiple autolysin or deoxyribonuclease activities directly in the gel as zones of clearing. Enzyme profiles of Proteobacteria which are, or were at one time, classified in the genus Pseudomonas were compared. For each species, a relatively large number of autolysin and deoxyribonuclease activities were detected. The distribution, numbers and intensities of zones of clearing in the gel provided complex species-specific patterns. Extensive data from two fundamental, and presumably evolutionarily distinct classes of enzymes were thus generated for purposes of comparison. Neither analysis suggested that these bacteria could represent a single natural cluster of species, lending support to their present multigeneric status. Ethidium-bromide-stained gels could be subsequently stained with Coomassie blue. This allowed the mapping of many deoxyribonuclease activities to particular peptides in the cell-free extract. In addition, modification of the substrate or renaturation buffer enabled a preliminary characterisation of several deoxyribonucleases in terms of their stability, substrate specificity, and other parameters expected to affect enzyme activity. Individual deoxyribonucleases could be located and screened for desired properties without prior purification.

Bacteria↗

PubMed2001

Unusual evolutionary history of the tRNA splicing endonuclease EndA: relationship to the LAGLIDADG and PD-(D/E)XK deoxyribonucleases.

The tRNA splicing endoribonuclease EndA from Methanococcus jannaschii is a homotetramer formed via heterologous interaction between the two pairs of homodimers. Each monomer consists of two alpha/beta domains, the N-terminal domain (NTD) and the C-terminal domain (CTD) containing the RNase A-like active site. Comparison of the EndA coordinates with the publicly available protein structure database revealed the similarity of both domains to site-specific deoxyribonucleases: the NTD to the LAGLIDADG family and the CTD to the PD-(D/E)XK family. Superposition of the NTD on the catalytic domain of LAGLIDADG homing endonucleases allowed a suggestion to be made about which amino acid residues of the tRNA splicing nuclease might participate in formation of a presumptive cryptic deoxyribonuclease active site. On the other hand, the CTD and PD-(D/E)XK endonucleases, represented by restriction enzymes and a phage lambda exonuclease, were shown to share extensive similarities of the structural framework, to which entirely different active sites might be attached in two alternative locations. These findings suggest that EndA evolved from a fusion protein with at least two distinct endonuclease activities: the ribonuclease, which made it an essential "antitoxin" for the cells whose RNA genes were interrupted by introns, and the deoxyribonuclease, which provided the means for homing-like mobility. The residues of the noncatalytic CTDs from the positions corresponding to the catalytic side chains in PD-(D/E)XK deoxyribonucleases map to the surface at the opposite side to the tRNA binding site, for which no function has been implicated. Many restriction enzymes from the PD-(D/E)XK superfamily might have the potential to maintain an additional active or binding site at the face opposite the deoxyribonuclease active site, a property that can be utilized in protein engineering.

Binding Sites↗

PubMed1989

Preparation of immobilized monomeric actin and its use in the isolation of protease-free and ribonuclease-free pancreatic deoxyribonuclease I.

A procedure is described for the immobilization of monomeric actin so that about 30% of the immobilized protein is competent to bind the monomeric-actin-binding proteins bovine pancreatic deoxyribonuclease I and chicken villin. The intact tertiary structure of the immobilized actin is required to bind these proteins. Using this resin, a method has been developed for the affinity purification of pancreatic deoxyribonuclease I on a reusable actin column. It involves the binding of deoxyribonuclease I to immobilized actin, extensive washing of the column, followed by elution of the bound deoxyribonuclease I with 10 M formamide. After removal of the formamide, the deoxyribonuclease I has a higher specific activity than the starting material and contained no detectable protease or ribonuclease contamination. This preparation should find considerable application in molecular genetic studies where the enzyme is needed free of these particular contaminants. The affinity column should also be useful for the isolation of other, physiologically relevant, monomeric-actin-binding proteins.

Actins↗

PubMed1972

Coagulase and deoxyribonuclease activities of staphylococci isolated from clinical sources.

A total of 504 clinical isolates of the family Micrococcaceae were tested for coagulase, deoxyribonuclease, clumping factor, and phosphatase to determine whether there is a correlation between the results of these tests and the pathogenicity of staphylococci. In the tests for coagulase production, it was found that either human or rabbit plasma could be used with broth cultures, whereas rabbit but not human plasma was satisfactory when microorganisms removed from solid culture medium were used. Deoxyribonuclease production correlated better than the fermentation of mannitol with coagulase production. The use of methyl green, Toluidine Blue O, or acridine orange offered no advantage over the use of HCl for detecting the production of deoxyribonuclease. Neither the presence of clumping factor nor the production of phosphatase correlated well with coagulase production. Strains of staphylococci that did not produce coagulase and deoxyribonuclease were isolated as frequently as, and from a greater variety of clinical sources than, strains which produced these substances. It is concluded that the production of coagulase and deoxyribonuclease are properties of staphylococci which are not necessarily indicative of potential pathogenicity of the organisms for man.

Acridines↗

PubMed1969

Extracellular deoxyribonuclease production by yeasts.

A total of 20 genera of yeasts and yeastlike organisms were tested for their ability to produce an extracellular deoxyribonuclease. Results indicate that ability to produce the enzyme appears to be a specific characteristic of the three genera Rhodotorula, Cryptococcus, and Tremella. A single strain of Endomycopsis fibuligera was also shown to be positive for the enzyme. In comparing the ability of the organisms to excrete extracellular deoxyribonuclease with their ability to produce urease, a surprisingly close correlation was found. With the exception of Lipomyces starkeyi, all the organisms which were deoxyribonuclease-negative were also urease-negative. Of those organisms which were deoxyribonuclease-positive, only E. fibuligera was urease-negative. The ability of cryptococci to produce extracellular deoxyribonuclease is discussed in relation to the implication which this finding may have for the taxonomy and phylogeny of the genus.

Culture Media↗

PubMedDate not supplied

Characterization of different deoxyribonucleases in human lymphocytes.

Four groups of deoxyribonuclease activities from human lymphocytes have been characterized by deoxyribonuclease assay in DNA-containing polyacrylamide gels following their separation by disc-electrophoresis. All activities hydrolyse DNA endonucleolytically. One neutral deoxyribonuclease found in the cytoplasmic fraction prefers native or UV-irradiated DNA over denatured DNA as substrate and is a 5'-monoester former. Two groups of acid deoxyribonuclease activities are detectable in the nuclear fraction. Both are 3'-monoester formers. One is as well active with denatured DNA as with native DNA, the other one shows the same activity with native and UV-irradiated DNA but lower activity with denatured DNA. An alkaline deoxyribonuclease activity, also localized in the nucleus, is a 5'-monoester former, and prefers denatured or UV-irradiated DNA as substrate.

Cell Nucleus↗

PubMed1990

[Deoxyribonuclease activity in the blood serum of persons participating in liquidation of the effects of the accident at the Chernobyl nuclear power plant].

Deoxyribonuclease activity in blood serum was comparatively analyzed in 90 subjects who had been engaged in liquidation of consequences of the catastrophe at Chernobyl NPS in 1986, and in 55 normal donors. It was found that the mean value of deoxyribonuclease activity in the group of the liquidators was significantly lower as compared to that of donors. A stable decrease of activity of neutral deoxyribonuclease (DNAse I, pH 7.3) was detected in 18 and that of acid deoxyribonuclease (DNAse II) in 9 out of 90 subjects investigated. The anamnesis of most of the patients with lowered deoxyribonuclease activity has revealed transient leukopenia, decreased parameters of T-cellular immunity and phagocytic activity of neutrophils.

Accidents↗

PubMed1993

Measurement of deoxyribonuclease I activity in human tissues and body fluids by a single radial enzyme-diffusion method.

In the single radial enzyme-diffusion (SRED) method for assay of deoxyribonuclease I, a precisely measured volume of the enzyme solution is dispensed into a circular well in an agarose gel layer in which DNA and ethidium bromide are uniformly distributed. A circular dark zone is formed as the enzyme diffuses from the well radially into the gel and digests substrate DNA. The diameter of the dark circle of hydrolyzed DNA increases in size with time and correlates linearly with the amount of enzyme applied to the well. Thus, the SRED can be used for quantitation of deoxyribonuclease I with a limit of detection of 2 x 10(-6) unit. This corresponds to 1 pg of purified urine deoxyribonuclease I. We measured the deoxyribonuclease I activity of 17 different human tissues and body fluids from healthy donors. Urine samples showed the greatest activity, 6.0 +/- 2.2 kilo-units/g protein (mean +/- SD). Serum deoxyribonuclease I activity was 4.4 +/- 1.8 units/L.

Adult↗

PubMed1986

Barley aleurone layers secrete a nuclease in response to gibberellic Acid : purification and partial characterization of the associated ribonuclease, deoxyribonuclease, and 3'-nucleotidase activities.

Incubation of barley (Hordeum vulgare L. cv Himalaya) half-seeds with gibberellic acid enhances the secretion of ribonuclease and deoxyribonuclease from aleurone tissue (MJ Chrispeels, JE Varner 1967 Plant Physiol 42: 398-406; L Taiz, JE Starks 1977 Plant Physiol 60: 182-189). These activities were over 50-fold greater in medium of half-seeds incubated with gibberellic acid than in control medium. Ribonuclease and deoxyribonuclease activities initially appeared in the medium 24 to 48 hours after hormone induction and increased for up to 96 hours. Both activities had a pH optimum of 6.0 and a temperature optimum of 55 degrees C. When the medium from gibberellic acid-treated half-seeds was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis, the major ribonuclease and deoxyribonuclease activity bands comigrated. The two enzyme activities remained associated throughout a 2,700-fold purification employing ammonium sulfate fractionation, Heparin-Agarose affinity chromatography, and Reactive Blue 2-Agarose affinity chromatography. Also accompanying the ribonuclease and deoxyribonuclease activities throughout purification was the ability to hydrolyze the 3'-phosphoester linkage of 3'-AMP. The purified protein was composed of a single polypeptide with an apparent molecular weight of 36 kilodaltons as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. It is concluded that in response to gibberellic acid, barley aleurone tissue secretes a nuclease having ribonuclease, deoxyribonuclease, and 3'-nucleotidase activities.

Journal Article↗

PubMed1977

The use of a novel plate assay in a search for yeast mutants defective in deoxyribonucleases.

Methods by which the intracellular enzymes deoxyribonuclease, ribonuclease and protease can be assayed in whole colonies of Saccharomyces cerevisiae on agar plates are described. A search for mutants deficient in deoxyribonuclease has been carried out. Two types of mutant are described. One apparently fails to produce deoxyribonuclease, ribonuclease or protease on agar plates and the other apparently fails to produce deoxyribonuclease and ribonuclease.

Clinical Enzyme Tests↗

PubMed1994

Deoxyribonuclease activity in Echinococcus granulosus protoscoleces and hydatid fluid.

Deoxyribonuclease activity was detected in E. granulosus protoscoleces from sheep hydatid cysts by electrophoresis in agarose gels of DNA fragments obtained after incubation of integral DNA with a protoscoleces preparation. Preliminary characterization showed that deoxyribonuclease activity was optimal at neutral-alkaline pH, magnesium ions were required, and it was able to digest different types of DNA, making random cuts. Electrophoresis in DNA-containing sodium dodecylsulfate (SDS)-polyacrylamide gels indicated a relative molecular mass, under non-reducing conditions, of 32 kDa. Deoxyribonuclease activity was also found in sheep hydatid fluid. It shared optimal pH, ionic and substrate requirements with the enzyme from protoscoleces but had a higher relative molecular mass (40 kDa), the same as that of normal sheep serum deoxyribonuclease.

Animals↗

PubMed1987

Assessment of preferential cleavage of an actively transcribed retroviral hybrid gene in murine cells by deoxyribonuclease I, bleomycin, neocarzinostatin, or ionizing radiation.

Preferential cleavage induced by bleomycin, neocarzinostatin, or ionizing radiation in a transcribed cellular gene was evaluated through comparisons with deoxyribonuclease I. The glucocorticoid-inducible LTL gene (a hybrid viral gene derived from mouse mammary tumor virus DNA) previously described [Zaret, K. S., & Yamamoto, K. R. (1984) Cell (Cambridge, Mass.) 38, 29-38] served as the specific DNA target. A Southern blot analysis was used to specifically assess cleavage of the LTL gene in nuclei isolated from cells either treated or untreated with the synthetic glucocorticoid dexamethasone. Hypersensitivity of the gene to bleomycin or neocarzinostatin, which paralleled deoxyribonuclease I hypersensitivity, was evident only in nuclei isolated from dexamethasone-treated cells. Like deoxyribonuclease I, sites of dexamethasone-inducible drug hypersensitivity were coincident with the binding region for the glucocorticoid receptor found within the regulatory sequences of the LTL gene. In contrast, no hypersensitivity to ionizing radiation was evident. Although bleomycin and neocarzinostatin showed qualitatively similar preferences for the transcribed LTL gene, quantitative evaluations of damage to total cellular DNA by filter elution showed that the relative specificity of bleomycin for the hypersensitive region was much less than that of either deoxyribonuclease I or neocarzinostatin.

Animals↗

PubMed1974

Role of a deoxyribonuclease in the genetic transformation of Diplococcus pneumoniae.

Two steps in the uptake of DNA by Diplococcus pneumoniae were characterized by analyzing mutants defective in transformation. A strain deficient in the two major deoxyribonucleases of D. pneumoniae takes up DNA normally and converts it to single strands within the cell and oligonucleotide fragments outside the cell. Extracts of this strain contain a residual deoxyribonuclease that produces similar oligonucleotide fragments in vitro. This enzyme is missing in transformation-defective mutants blocked in the second or entry step. Cells of this mutant class bind large amounts of DNA to their surface in a form accessible to external agents. Another class of nontransformable mutants fails to bind DNA at all. Their deoxyribonuclease content is unchanged, and they are apparently blocked in the first or binding step of DNA uptake. The binding step requires a source of energy and prior activation of the cells by competence factor. Entry may be independent of these requirements and may come about by action of the deoxyribonuclease on one strand of DNA with energy for the transport of the intact strand deriving from hydrolysis of the degraded strand. The enzyme may thus act as a DNA translocase.

Binding Sites↗

PubMed1993

DNA-sequence binding preference of the GC-selective ligand mithramycin. Deoxyribonuclease-I/deoxyribonuclease-II and hydroxy-radical footprinting at CCCG, CCGC, CGGC, GCCC and GGGG flanked by (AT)n and An.Tn.

We have used hydroxy-radical and deoxyribonuclease-I footprinting to probe the interaction of mithramycin with DNA fragments containing the sequences (AT)10X(AT)10 (X = CCCG, CCGC or CGGC) and A14GCCCT15. As expected the drug produces clear footprints located around the central four GC base pairs. The exact position of the footprint is different for the four sequences; the footprint with CCCG is displayed by two base pairs in the 5' direction relative to GCCC. These variations are explained by suggesting that mithramycin avoids the dinucleotide CG and binds better to GG/CC than GC. Although there is little change in deoxyribonuclease-I cleavage of the surrounding blocks of (AT)n, cleavage by deoxyribonuclease II is markedly enhanced and certain thymines on the 5' side of the ligand-binding site become hyperreactive to hydroxy-radical attack. Adjacent regions of An.Tn show enhanced rates of deoxyribonuclease-I cleavage in the presence of the antibiotic.

DNA↗

PubMed1997

Effects of minor and major groove-binding drugs and intercalators on the DNA association of minor groove-binding proteins RecA and deoxyribonuclease I detected by flow linear dichroism.

Linear and circular dichroic spectroscopies have been employed to investigate the effects of small DNA ligands on the interactions of two proteins which bind to the minor groove of DNA, viz. RecA protein from Escherichia coli and deoxyribonuclease I (bovine pancreas). Ligands representing three specific non-covalent binding modes were investigated: 4',6-diamidino-2-phenylindole and distamycin A (minor groove binders), methyl green (major groove binder), and methylene blue, ethidium bromide and ethidium dimer (intercalators). Linear dichroism was demonstrated to be an excellent detector, in real time, of DNA double-strand cleavage by deoxyribonuclease I. Ligands bound in all three modes interfered with the deoxyribonuclease I digestion of dsDNA, although the level of interference varied in a manner which could be related to the ligand binding site, the ligand charge appearing to be less important. In particular, the retardation of deoxyribonuclease I cleavage by the major groove binder methyl green demonstrates that accessibility to the minor groove can be affected by occupancy of the opposite groove. Binding of all three types of ligand also had marked effects on the interaction of RecA with dsDNA in the presence of non-hydrolyzable cofactor adenosine 5'-O-3-thiotriphosphate, decreasing the association rate to varying extents but with the strongest effects from ligands having some minor groove occupancy. Finally, each ligand was displaced from its DNA binding site upon completion of RecA association, again demonstrating that modification of either groove can affect the properties and behaviour of the other. The conclusions are discussed against the background of previous work on the use of small DNA ligands to probe DNA-protein interactions.

Circular Dichroism↗