DNases of Acholeplasma spp

One strain from each of seven species of Acholeplasma was examined for the presence of DNases. Six of the strains were found to be DNase positive when assayed with DNA-methyl-green-containing agar, indicating that this method can be used to differentiate the seventh strain, Acholeplasma axanthum, from the remaining Acholeplasma spp. Electrophoretic patterns obtained from sodium dodecyl sulfate-polyacrylamide gels containing DNA revealed DNases in the cell extracts of all seven strains and in the supernatant growth medium of five of the strains. The electrophoretic patterns were highly characteristic for each strain and can be used for the rapid identification of different strains of Acholeplasma.     

Isoelectric focusing of multiple forms of DNase in thin layers of polyacrylamide gel and detection of enzymatic activity with a zymogram method following separation

Multiple forms of bovine pancreatic DNase (DNases A, B, C, and D) are separated by isoelectric focusing in thin layers of polyacrylamide gel with a carrier ampholyte in the pH range 4–6. The isoelectric points of DNases A, B, C, and D are 5.22, 4.96, 5.06, and 4.78, respectively. A zymogram method for detecting DNase activity as bands in the gel following isoelectric focusing is described. The method detects microgram amounts of DNase and has only one step. It can be used with the parified cazyme as well as with crude extracts of tissues containing DNase. By this method, two major components of DNase in ovine pancreas and at least three in malted barley as well as two previously unideatified forms of DNase in bovine pancreas with isoelectric points of 5.12 and 5.48 (DNases E and F) are observed.     

Possible Relationship Between Yeast Deoxyribonucleases and Deoxyribonucleic Acid Yield

The deoxyribonucleic acid (DNA) yield and deoxyribonuclease (DNase) activity of several yeasts were correlated. Debaryomyces castellii and Debaryomyces franciscae were found to contain active DNases which carry out DNA hydrolysis, whereas the amounts of DNA as determined by extraction with Sarkosyl buffer (pH 7.8) were found to be small. On the other hand, Candida parapsilosis, Saccharomyces carmosousae, and Lodderomyces elongisporus produced no detectable DNases active at pH 7.8, and their DNA yield was correspondingly high. L. elongisporus was found to possess DNase only at pH 4.0.     

Isolation and characterization of multiple forms of malt deoxyribonuclease

A deoxyribonuclease (DNase), similar to bovine pancreatic DNase, has been isolated from germinating barley. Commerically available malt was used as source of the enzyme. The purification procedure involves (a) ammonium sulfate fractionation (45–65% saturation), (b) CM-cellulose chromatography at pH 4.7 and (c) DEAE-cellulose chromatography at pH 8. DEAE-cellulose separates the enzyme into 4 distinct forms, designed as DNases A, B, C, and D. DNase A and B may be rechromatographed on DEAE-cellulose employing a CaCl₂ instead of Tris-HCl gradient. Both forms appear homogeneous on regular and sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. In addition, both forms have a sp. act. of ca 700 units per A unit at 280 nm, similar to the potency of the pancreatic enzyme. DNase C and D, which are present in relatively small quantities in malt, were not characterized. The MWs of DNases A and B, as estimated by the SDS gel electrophoresis techniques, are near 32 000, slightly larger than that of the pancreatic enzyme. In the presence of either Mn²⁺ or Mg²⁺, the pH-activity profile of the barley enzyme is similar to that obtained with the pancreatic enzyme. Like the pancreatic enzyme, barley DNase is protected by Ca²⁺ from inactivation. The amino acid compositions of the A and B forms are about the same; a comparison of the malt and pancreatic enzymes shows many similarities but major differences in the amounts of glutamic acid, proline and glycine. The hydrolysis products of DNA by malt DNase are indistinguishable from those obtained with pancreatic DNase. Further hydrolysis of these products by snake venom phosphodiesterase shows malt DNase to be a 5′-phosphate producer. Deoxythymidine 3′,5′-di-p-nitrophenyl phosphate, one of the synthetic substrates of pancreatic DNase, is also hydrolysed by malt DNase.     

Experimental and computational analyses of the energetic basis for dual recognition of immunity proteins by colicin endonucleases

Colicin endonucleases (DNases) are bound and inactivated by immunity (Im) proteins. Im proteins are broadly cross-reactive yet specific inhibitors binding cognate and non-cognate DNases with K_d values that vary between 10^− 4 and 10^− 14 M, characteristics that are explained by a ‘dual-recognition’ mechanism. In this work, we addressed for the first time the energetics of Im protein recognition by colicin DNases through a combination of E9 DNase alanine scanning and double-mutant cycles (DMCs) coupled with kinetic and calorimetric analyses of cognate Im9 and non-cognate Im2 binding, as well as computational analysis of alanine scanning and DMC data. We show that differential ΔΔGs observed for four E9 DNase residues cumulatively distinguish cognate Im9 association from non-cognate Im2 association. E9 DNase Phe86 is the primary specificity hotspot residue in the centre of the interface, which is coordinated by conserved and variable hotspot residues of the cognate Im protein. Experimental DMC analysis reveals that only modest coupling energies to Im9 residues are observed, in agreement with calculated DMCs using the program ROSETTA and consistent with the largely hydrophobic nature of E9 DNase-Im9 specificity contacts. Computed values for the 12 E9 DNase alanine mutants showed reasonable agreement with experimental ΔΔG data, particularly for interactions not mediated by interfacial water molecules. ΔΔG predictions for residues that contact buried water molecules calculated using solvated rotamer models met with mixed success; however, we were able to predict with a high degree of accuracy the location and energetic contribution of one such contact. Our study highlights how colicin DNases are able to utilise both conserved and variable amino acids to distinguish cognate from non-cognate Im proteins, with the energetic contributions of the conserved residues modulated by neighbouring specificity sites.     

Deoxyribonuclease Inhibitory Activity in Pig Intestinal Contents

The inhibitory effect of pig intestinal contents on some microbial DNases and bovine pancreas DNase was examined employing an agar diffusion test. The molecular weight of 1 inhibitor as well as the electrophoretic patterns of the inhibitors were determined. DNases from Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pyogenes, Clostridium perfringens and bovine pancreas were inhibited by intestinal contents. DNases from Clostridium septicum, Serratia marcescens, Proteus mirabilis, Aeromonas hydropnila and Pseudomonas aeruginosa were not inhibited. The concentrations of the inhibitory substances were considerably higher in contents from the small intestine than from the large intestine. Using electrophoretic procedures on intestinal contents, 3 different fractions showing DNase inhibition were demonstrated. The molecular weight of one of the inhibitors was estimated to be about 30000.     

Deoxyriboendonucleases with exocytoplasmic location in Streptomyces

Summary DNase activities that show a non-specific pattern of DNA degradation on agarose gels have been detected in six strains of Streptomyces. All the enzymes were located in the cell wall-cytoplasmic membrane space, and released after protoplast production. Synthesis of these nucleases was dependent on the composition of the growth media. Differentiation on agar plates was affected by the same nutritional conditions that regulate DNAse production. All DNases were deoxyriboendonucleases that required Mg²⁺ and a low ionic concentration for optimal activity. The molecular masses of native DNases range from 37±2 to 43±4 kDa. The enzymes produced nicks in double-stranded DNA and were unable to digest RNA. The widespread presence, location and characteristics of such nucleases in Streptomyces suggest a common role for these enzymes. Offprint requests to: J. Sánchez     

Deoxyribonucleases (DNases) in the cortex and endosome from the marine sponge <Emphasis Type="Italic">Tethya aurantium</Emphasis>

The presence and activity of deoxyribonucleases in the cortex and endosome sections from a sponge, the sea orange Tethya aurantium, were investigated. The maximal enzyme activity in sponge homogenate was detected at pH 4.27, pH 7.0 and pH 8.5–8.75. Among different specimens, several distinct patterns of neutral DNase isozymes were observed in the cortex section. In each investigated specimen the highest neutral DNase activity belonged to high molecular weight proteins (up to75 kDa). The acid DNases showed a low level of enzyme activity. In the endosome section the acid DNase activity was up to ten times higher than in the cortex and the presence of DNase II-like protein was detected. Neutral DNase, which expressed the highest enzyme activity in all the investigated specimens, has a molecular weight of 20 kDa and belongs to the DNase I-like family. The results indicate that the activity of neutral and acid DNases is related to sponge sections and their biological functions. The cortex, as the sponge section that communicates with the environment, expresses high interindividual variability and heterogeneity of neutral DNases, while the endosome section, where the intracellular digestion is localized, is a site of high acid DNase activity.     

Interaction of Nuclease Colicins with Membranes: Insertion Depth Correlates with Bilayer Perturbation

Background

Protein transport across cellular membranes is an important aspect of toxin biology. Escherichia coli cell killing by nuclease colicins occurs through DNA (DNases) or RNA (RNases) hydrolysis and to this end their cytotoxic domains require transportation across two sets of membranes. In order to begin to unravel the molecular mechanisms underlying the membrane translocation of colicin nuclease domains, we have analysed the membrane association of four DNase domains (E9, a charge reduction E9 mutant, E8, and E7) and one ribosomal RNase domain (E3) using a biomembrane model system.

Principal Results

We demonstrate, through the use of large unilamellar vesicles composed of synthetic and E. coli lipids and a membrane surface potential sensor, that the colicin nuclease domains bind anionic membranes only, with micromolar affinity and via a cooperative binding mechanism. The evaluation of the nuclease bilayer insertion depth, through a fluorescence quenching analysis using brominated lipids, indicates that the nucleases locate to differential regions in the bilayer. Colicin DNases target the interfacial region of the lipid bilayer, with the DNase E7 showing the deepest insertion, whereas the ribosomal RNase E3 penetrates into the hydrophobic core region of the bilayer. Furthermore, the membrane association of the DNase E7 and the ribosomal RNase E3 induces vesicle aggregation, lipid mixing and content leakage to a much larger extent than that of the other DNases analysed.

Conclusions/Significance

Our results show, for the first time, that after the initial electrostatically driven membrane association, the pleiotropic membrane effects induced by colicin nuclease domains relate to their bilayer insertion depth and may be linked to their in vivo membrane translocation.     

The Applicability of Agar-Diffusion Test in Measuring Deoxyribonucleases in Pig Intestinal Content

The suitability of an agar-diffusion test (ADT) using toluidine blue deoxyribonucleic acid agar (TDA) for measuring DNase activity in pig intestinal contents was investigated. The ADT was compared with a spectrophotometrical method. Distinct metachromatic zones around wells in the DNA-containing agar, into which the intestinal content was applied, indicated DNase activity. The DNase activity was determined semiquantitatively by making serial twofold dilutions of the intestinal content. The spectrophotometrical method was optimal at pH 7.2. The ADT proved to be most sensitive at pH 5.6. The ability of the 2 methods employed to measure low concentrations of DNases was equal. However, the ADT was considered more suitable than the spectrophotometrical method because ADT measured reduced amounts of enzyme. DNase activity was demonstrated throughout the small intestine and in the large intestine. By the zymogram technique, at least 3 different DNases could be demonstrated in the lower parts of the small intestine, 1 of which could be of extrapancreatic origin.     

Glucocorticoid hormones are successively present in two sites with different accessibilities to nucleases in chromatin from HTC cells

Glucocorticoid hormones induce tyrosine aminotransferase synthesis in HTC cells after a lag of approximately 2 h. The question arises whether this lag corresponds to chromatin modifications related to the binding of the hormone. We have analyzed the accessibility to DNases of the hormone binding sites in chromatin from HTC cells incubated for various times in the presence of the hormone. After short incubations the hormone-receptor binding sites in chromatin are accessible to DNases, after longer incubations they become resistant to DNase digestion. The kinetics of the release in vivo of the hormone-receptor complex in an hormone free medium suggests the existence of 2 discrete classes of hormone binding sites in chromatin.     

DNases and apoptosis.

Here we review the different apoptotic DNases. From a functional point of view, DNases implicated in apoptosis may be classified into three groups: the Ca2+/Mg2+ endonucleases, the Mg2+-endonucleases, and the cation-independent endonucleases. The first group includes DNase I which has no specificity for the linker region, DNase gamma which has some homology with DNase I, and other DNases which cleave DNA in the linker region. Both DNase I and DNase gamma have been cloned. The other nucleases of this category have dispersed molecular weights. Their sequences are unknown and it is difficult to determine their role(s) in apoptosis. It seems that different pathways are present and that these nucleases may be activated either by caspases or serine proteases. The caspase 3 activated DNase (CAD, CPAN, or DFF40) belongs to the Mg2+-dependent endonucleases. DNase II belongs to the third group of acid endonucleases or cation-independent DNases. We have shown the involvement of DNase II in lens cell differentiation. Recently, the molecular structure of two different enzymes has been elucidated, one of which has a signal peptide and appears to be secreted. The other, called L-DNase II, is an intracellular protein having two enzymatic activities; in its native form, it is an anti-protease, and after posttranslational modification, it becomes a nuclease.     

The immunological and structural comparisons of deoxyribonucleases I. Glycosylation differences between bovine pancreatic and parotid deoxyribonucleases

A rabbit antiserum against bovine pancreatic DNase A is used to study the immunological reaction of DNases I. As shown by double immunodiffusion, bovine pancreatic DNases A, B, C, and D are immunologically identical, so are DNases from bovine pancreas and parotid and from ovine pancreas. These DNases also behave similarly in immunotitration of DNase activity and all are tightly bound to the immunoaffinity medium, requiring an acidic buffer with 10% ammonium sulfate to dissociate. On the other hand, porcine pancreatic and malted barley DNases that do not form precipitin lines remain active in solution with the antibody; however, in spite of the lack of inhibition these DNases are retarded (but not tightly bound) in immunoaffinity chromatography, suggesting interaction with the antibody. In thin layer isoelectric focusing, the parotid DNase, purified with the immunoaffinity technique, shows only two major active components whose isoelectric points correspond to those of DNases A and C of bovine pancreas. As estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the molecular weight of parotid DNase is 34,000, approximately 3,000 more than that of the pancreatic enzyme. However, both parotid and pancreatic DNases have the same NH2-terminal leucine, an identical COOH-terminal amino acid sequence, nearly identical amino acid compositions, and almost the same peptide maps. The molecular weight difference is due to differences in the carbohydrate side chains. Results of peptide analyses indicate that parotid DNase contains two glycopeptides; pancreatic DNase has only one. In addition, both parotid glycopeptides contain glucosamine and galactosamine while the pancreatic glycopeptide has only glucosamine.     

Deoxyribonucleases from rat brain

Two distinctly different DNases were isolated from rat brain and could be separated easily by ammonium sulphate fractionation. One of the DNases acts optimally at pH 5.0 hydrolysing preferentially native DNA and requiring an optimal Mg²⁺ concentration of about 0.03 m . The other DNase has its optimal pH between 7.4 and 8.9, acts preferentially on heat-denatured DNA and requires a lower Mg²⁺ concentration, the optimum being 1 × 10^?4m . Cerebellum from adult rat brain has a lower acid DNase activity and higher alkaline DNase activity, and therefore has a higher ratio of alkaline DNase to acid DNase than the other areas of brain studied. This unique activity ratio in cerebellum of adult rat brain was not observed in infant rat brain.     

Endonuclease activities in the coleoptile and the first leaf of developing etiolated wheat seedlings

DNase activity in coleoptiles and the first leaf apices of winter wheat (Triticum aestivum L., cv. Mironovskaya 808) etiolated seedlings was found to increase significantly during seedling growth, peaking on the eighth day of plant development. The maximum of DNase activity was coincident with apoptotic internucleosomal DNA fragmentation in these organs. Wheat endonucleases are capable of hydrolyzing both singleand double-stranded DNA of various origins. The leaf and coleoptiles were found to exhibit nuclease activities that hydrolyzed the lambda phage DNA with N⁶-methyladenine and 5-methylcytosine more actively compared to the hydrolysis of similar unmethylated DNAs. Thus, the endonucleases of wheat seedlings are sensitive to the methylation status of their substrate DNAs. The leaves and coleoptiles exhibited both Ca²⁺/Mg²⁺- and Zn²⁺-dependent nuclease activities that underwent differential changes during development and senescence of seedling organs. EDTA at a concentration of 50 mM fully inhibited the total DNase activity. Electrophoretic heterogeneity was observed for DNase activities operating simultaneously in the coleoptile and the first leaf at different stages of seedling development. Proteins exhibiting DNase activity (16–80 kD mol wt) were revealed in the first leaf and the coleoptile; these proteins were mostly nucleases with the pH optimum around 7.0. Some endonucleases (mol wts of 36, 39, and 28 kD) were present in both organs of the seedling. Some other DNases (mol wts of 16, 56, and about 80 kD) were found in the coleoptile; these DNases hydrolyzed DNA in the nucleus at terminal stages of apoptosis. Different suites of DNase activities were revealed in the nucleus and the cytoplasm, the nuclear DNase activities being more diverse than the cytoplasmic ones. Thus, the cellular (organspecific) and subcellular heterogeneity in composition and activities of DNases has been revealed in wheat plants. These DNases undergo specific changes during seedling development, serving at various stages of programmed cell death in seedling tissues.     

Mutagenic scan of the H-N-H motif of colicin E9: implications for the mechanistic enzymology of colicins,homing enzymes and apoptotic endonucleases

Colicin E9 is a microbial toxin that kills bacteria through random degradation of chromosomal DNA. Within the active site of the cytotoxic endonuclease domain of colicin E9 (the E9 DNase) is a 32 amino acid motif found in the H-N-H group of homing endonucleases. Crystal structures of the E9 DNase have implicated several conserved residues of the H-N-H motif in the mechanism of DNA hydrolysis. We have used mutagenesis to test the involvement of these key residues in colicin toxicity, metal ion binding and catalysis. Our data show, for the first time, that the H-N-H motif is the site of DNA binding and that Mg²⁺-dependent cleavage of double-stranded DNA is responsible for bacterial cell death. We demonstrate that more active site residues are required for catalysis in the presence of Mg²⁺ ions than transition metals, consistent with the recent hypothesis that the E9 DNase hydrolyses DNA by two distinct, cation-dependent catalytic mechanisms. The roles of individual amino acids within the H-N-H motif are discussed in the context of the available structural information on this and related DNases and we address the possible mechanistic similarities between caspase-activated DNases, responsible for the degradation of chromatin in eukaryotic apoptosis, and H-N-H DNases.     

Extracellular DNase activity of Cryptococcus neoformans and Cryptococcus gattii

Extracellular DNase activity was studied in 73 strains of Cryptococcus neoformans and 12 strains of Cryptococcus gattii. DNase activity was measured by DNase agar clearance with and without Methyl Green. All strains tested showed extracellular DNase activity and no significant difference was found betweenC. neoformans and C. gattii strains. DNase production was higher in strains from clinical origin (average radius of 6.2 mm) than among environmental strains (average radius of 2.9 mm). The extracellular enzyme may be detected by DNA substrate PAGE assays and its molecular weight was estimated at 31 kD. These results suggest that extracellular DNase could be considered as a virulence factor involved in C. neoformans–C. gattii species complex pathogenicity.     

Non-classic characteristics define prominent DNase activities from the intestine and other tissues of Haemonchus contortus

The anthelmintic fenbendazole (FBZ) induces nuclear DNA fragmentation (DF) in intestinal cells of Haemonchus contortus. The DNA fragments had 3'-OH, which suggests involvement of a neutral DNase. To identify candidate DNase(s) involved, DNase activity in H. contortus intestine and other worm fractions was characterized relative to classic DNases I (neutral) and II (acidic). Seven distinct DNase activities were identified and had Mrs of 34, 36, 37 or 38.5 kDa on zymographic analysis. The different activities were distinguished according to pH requirement, sensitivity to 10 mM EDTA and worm compartment. Activities of intestinal DNases at 34, 36 and 38.5 kDa were sensitive to EDTA at pH 5.0 and 7.0. Sensitivity to EDTA at pH 5.0 was unexpected compared to classic acidic DNase II activity, suggesting unusual properties of these DNases. In whole worms, however, the activities at 36 and 38.5 kDa were relatively insensitive to EDTA, indicating predominance of DNases that are distinct from the intestine. The activity at 37 kDa in excretory/secretory products had an acidic pH requirement and was insensitive to EDTA, resembling classic acidic DNase activity. Under conditions of pH 5.0 and 7.0, intestinal DNases produced 3'-ends that could be labeled by terminal deoxynucleotidyl transferase, indicating presence of 3'-OH. The labeling of 3'-ends at pH 5.0, again, was unexpected for acidic DNase activity. These results and several other activities suggest that multiple H. contortus DNases have characteristics distinct from the classic mammalian DNases I and II. Treatment of H. contortus with FBZ did not induce any detectable DNase activities distinct from normal intestine, although relative activities of intestinal DNases appear to have been altered by this treatment.     

The Demonstration and Characterization of Deoxyribonucleases of Streptococci Group A,B, C,G And L

Streptococcus pyogenes, S. agalactiae, S. dysgalactiae, S. equi, S. equisimilis, S. zooepidemicus, Streptococcus group G and L were found to produce deoxyribonucleases (DNases) which were demonstrated using the Toluidine Blue DNA Agar (TDA) described for staphylococcal DNases. The activity of streptococcal DNases increased in the presence of Mg++ and Ca++ ions, the pH optimum was about 7.5 and native DNA was the best enzyme substrate. It is consequently recommended to modify the TDA according to these results for the demonstration of streptococcal DNases. All streptococcal DNases, except the DNase of S. zooepidemicus, were found to be heat-stable. Isoelectric focusing was a convenient technique for separation of streptococcal DNases and for estimation of the pI values of the DNases. S. agalactiae and S. dysgalactiae generally exhibited distinct species specific patterns in the isoelectric focusing experiments. The DNases produced by S. pyogenes were serologically related to the DNases of S. dysgalactiae and Streptococcus group G. A similar relationship was demonstrated between the DNases produced by S. equisimilis and Streptococcus group L.     

Single-step Purification by Lectin Affinity and Deglycosylation Analysis of Recombinant Human and Porcine Deoxyribonucleases I Expressed in COS-7 Cells

Human and porcine recombinant deoxyribonucleases I (DNases I) were expressed in COS-7 cells, and purified by a single-step procedure. Since affinities for concanavalin A (Con A) and wheatgerm agglutinin (WGA) were strong in these recombinant DNases I, purification using Con A–WGA mixture-agarose column was performed. By this method, the enzymes in culture medium could quickly be isolated to apparent homogeneity in approx. 10 min. From 1 ml of culture medium, about 20–30 μg of purified DNase I with a specific activity ranging from 22000 to 41000 units/mg were obtained. The purified DNases I were subjected to enzymatic deglycosylation by either peptide N-glycosidase F (PNGase F) or endoglycosidase H (Endo H). The recombinant enzyme was cleaved by PNGase F, but not by Endo H, indicating that the recombinant enzymes are modified by N-linked complex-type carbohydrate moieties. In the human recombinant DNase I, activity was decreased by PNGase F-treatment, while that of the porcine DNase I remained unaffected. The thermal stability of the human enzyme was extremely susceptible to heat following PNGase F-treatment, as was the porcine enzyme to a lesser extent. This study suggests that N-linked complex-type carbohydrate moieties may contribute to the enzymatic activity and/or thermal stability of recombinant DNases I.