An endonuclease fromCaenorhabditis elegans: Partial purification and characterization

A deoxyribonuclease was partially purified from the free-living nematodeCaenorhabditis elegans. The DNase functioned as an endonuclease and introduced both single-strand nicks and double-strand breaks into DNA. The enzyme hydrolyzed double-stranded DNA seven times more rapidly than single-stranded DNA. DNase activity was not affected by the addition of divalent cations below 1mm but was inhibited at higher ionic concentrations. In addition, the enzyme was not inhibited in the presence of 10mm EDTA. The enzyme was inhibited by salt concentrations greater than 20mm. Three independent mutations in thenuc-1 gene were shown to reduce nuclease activity to less than 1% of that seen in wild-type organisms. This work was supported by National Institutes of Health Grant AG03161 and a TCU Research Foundation Grant. Some stocks used in these experiments were obtained from theCaenorhabditis Genetics Center, which is supported by Contract NOI-AG-9-2113 between the NIH and the curators of the University of Missouri.     

Purification and Properties of Deoxyribonuclease B From Bacillus subtilis

An exonuclease, DNase B, was isolated from Bacillus subtilis Marburg strain. Molecular weight of DNase B was estimated to be 200,000 by glycerol gradient centrifugation, however, 56,000 by SDS-polyacrylamide gel electrophoresis. This result would indicate a possibility that the enzyme consisted of an identical subunit. The enzyme was specific for single-stranded DNA, required Mg²⁺ or Mn²⁺ (5 mm) for the maximal activity, but 40% of the activity was retained in the absence of divalent cations. The enzyme was active even in the presence of EDTA. The enzyme degraded single-stranded DNA exonucleolytically, producing oligonucleotides in the direction from the 5′-end to the 3′-end.     

An enzyme from the earthworm Eisenia fetida is not only a protease but also a deoxyribonuclease

The earthworm enzyme Eisenia fetida Protease-III-1 (EfP-III-1) is known as a trypsin-like protease which is localized in the alimentary canal of the earthworm. Here, we show that EfP-III-1 also acts as a novel deoxyribonuclease. Unlike most DNases, this earthworm enzyme recognizes 5′-phosphate dsDNA (5′P DNA) and degrades it without sequence specificity, but does not recognize 5′OH DNA. As is the case for most DNases, Mg²⁺ was observed to markedly enhance the DNase activity of EfP-III-1. Whether the earthworm enzyme functioned as a DNase or as a protease depended on the pH values of the enzyme solution. The protein acted as a protease under alkaline conditions whereas it exhibited DNase activity under acid conditions. At pH 7.0, the enzyme could work as either a DNase or a protease. Given the complex living environment of the earthworm, this dual function of EfP-III-1 may play an important role in the alimentary digestion of the earthworm.     

Purification and properties of an acid deoxyribonuclease from rat small intestinal mucosa

An acid deoxyribonuclease has been purified from rat small intestinal mucosa by a procedure including ammonium sulfate fractionation, chromatographies on DEAE-cellulose, CM-cellulose and SE-Sephadex and finally isoelectric focusing. Polyacrylamide gel electrophoresis of the purified enzyme preparation showed one major and two minor bands, and the enzyme activity corresponded to one of the minor bands. The enzyme preparation was free of contaminating DNase I, DNase III, alkaline RNase, acid and alkaline phosphatases and nonspecific phosphodiesterase, but slight activities of DNase IV and acid RNase were detected. The enzyme did not require divalent cations for activity, had a pH optimum of 4.5 in 0.33 M sodium acetate buffer, and had an optimum temperature of 50 to 60 degrees C when assayed for 30 min. The rate of hydrolysis of native DNA was about 2.5-fold faster than that observed with denatured DNA. Its molecular weight was found to be 9.0 +/- 0.1. The enzyme catalyzes the endonucleolytic cleavage of native and denatured DNA, yielding oligonucleotides which have an average chain length of about 7, and which contain 3'-phosphoryl termini. The mode of action of the enzyme is double-strand scission.     

Human urine DNase I: immunological identity with human pancreatic DNase I, and enzymic and proteochemical properties of the enzyme

DNase I in human urine was purified to an electrophoretically homogeneous state by column chromatographies on DEAE-lignocellulose, hydroxyapatite, DEAE-cellulose, Sephadex G-75 and elastin-celite. The purified enzyme was immunologically identical with human pancreatic DNase I, but not with bovine pancreatic DNase I. The molecular weight and isoelectric point of the enzyme were estimated to be 4.1 X 10(4) and 3.6, respectively. The amino acid analysis revealed that 1 mol of the enzyme contained 8 mol of half-cystine. The N-terminal amino acid was identified as leucine by the dansyl chloride method. The enzyme was active in the presence of Mg2+, Co2+, or Mn2+, The optimum pH was around 6.5. The enzyme was stable in the pH range from 5.0 to 9.0 and at temperatures lower than 45 degrees C. The rate of hydrolysis of native DNA by the enzyme was twice as fast as that observed with heat-denatured DNA. This enzyme exhaustively degraded about 20% of the phosphodiester bonds in native DNA. The enzyme also degraded poly(dA) and poly(dT), but hardly degraded poly(dG) and poly(dC).     

Deoxyribonuclease II from the Icelandic scallop (Chlamys islandica): isolation and partial characterization

A deoxyribonuclease (DNase) was isolated from viscera of the cold-adapted marine bivalve Icelandic scallop. The 42 kDa DNase was shown to be a single polypeptide which catalyses DNA hydrolysis in the absence of divalent cations. The isolated enzyme showed maximal activity at pH 6 and no activity above pH 7.2 against native DNA. The scallop DNase was slightly more susceptible to heat denaturation than porcine DNase II and makes double-strand breaks in circular DNA substrate as the porcine enzyme. The N-terminal sequence of the scallop DNase was shown to be closely similar to DNase II (EC 3.1.22.1) proteins from other organisms. The scallop DNase is in addition to plancitoxin I from A. planci, the only DNase II enzyme isolated from marine invertebrates.     

A single,phosphate-repressible deoxyribonuclease,DNase A,secreted inAspergillus nidulans

High levels of nuclease activities were identified in filtrates ofAspergillus cultures after growth in low- but not in high-phosphate media. Deoxyribonuclease activities, characterized extensively by column chromatography, showed a coincident single peak for ss- and ds-DNase which was distinct from the peak for RNase. Both ss-DNase and ds-DNase are endonucleolytic and showed the highest activity in the presence of Ca²⁺ and Mn²⁺ (atpH 8.0). They also showed identical heat sensitivities suggesting that a single, phosphate-repressible DNase was secreted. This enzyme, therefore, corresponds to the well-characterized extracellular DNase A ofNeurospora. However, theAspergillus DNase A did not cross-react with antisera to secretedNeurospora nucleases and showed different chromatographic properties, and active peptides of different sizes were visualized on DNA activity gels. The increasing derepression ofAspergillus DNase A by decreasing phosphate levels was similar to that of secreted alkaline phosphatase and these increases were both abolished by the regulatory mutantpalcA. This investigation was supported by Grant A2564 from the Natural Science and Engineering Research Council of Canada.     

Purification and some properties of deoxyribonuclease whose synthesis is controlled by gene 49 of bacteriophage T4.

An enzyme which specifically cleaves very-fast-sedimenting DNA of bacteriophage T4 is synthesized after infection of T4, and its synthesis is controlled by gene 49 [1,2]. This enzyme has been proved to be a DNase [2]. We have purified this DNase 3000-fold from extracts of E. coli infected with T4. The purified preparation was practically free from other DNases, and the DNase activity was not detectable in cells infected with a mutant defective in gene 49. The enzyme activity from cells infected with a temperature-sensitive mutant of gene 49 was also temperature-sensitive, suggesting strongly that gene 49 is a structural gene of the DNase. The molecular weight of the wild-type enzyme was estimated to be 50 x 10(3) by gel filtration chromatography. The purified DNase did not cleave native and denatured DNAs of T3 and T4, but cleaved renatured T3 DNA with enzymatically fragmented T3 DNA, indicating that gaps in the DNA duplex are structures susceptible to the DNase. Cleavage of the hybridized T3 DNA occurred when the fragmented DNA was phosphorylated at either the 3' or 5'-strand termini.     

Carp liver DNase--isolation, further characterization and interaction with endogenous actin

Deoxyribonuclease I (DNase I)-like enzyme from the liver of the carp (Cyprinus carpio) was purified to homogeneity and further characterized. Ion exchange chromatography on DEAE-cellulose, molecular filtration on Sephacryl S-300 and Con A-Sepharose affinity chromatography were applied for enzyme isolation. Carp liver DNase, similarly to DNase I from bovine pancreas, was found to be an endonuclease that hydrolyses linear DNA from salmon sperm as well as circular DNA forms--plasmid and cosmid. The purified enzyme is a glycoprotein and shows microheterogeneity, as observed in DNase zymograms prepared after native and two-dimensional electrophoresis (2D-PAGE). The composition of sugar component of the enzyme was characterized. Special attention was focused on the ability of carp liver DNase to interact with carp liver actin. The carp liver enzyme was inhibited by endogenous actin. The estimated binding constant of carp liver DNase to carp liver actin was calculated to be 1.1 x 10(6) M(-1).     

Does DNA absorb microwave energy?

The microwave absorption of chicken erythrocytes and E. coli DNA aqueous solutions was studied in the 9–12-GHz frequency range by the method of variable thickness. At the same frequencies, fragments of sonicated erythrocyte DNA (average size about 500 base pairs) and of E. coli DNA treated with DNase (most of which were 800–2800 base pairs) were investigated. In neither case was any effect of enhanced microwave absorption by DNA observed. It was shown that an excess absorption of DNA solution falls within the 1% experimental error range, provided the conductivity contribution of 1% MgCl₂, required for DNase action, to the microwave absorption is taken into account.     

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.     

DNase I sensitive domain of the gene coding for the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase

We have cloned a 36-kilobase segment of chicken DNA containing the gene coding for glyceraldehyde-3-phosphate dehydrogenase [GAPDH (EC 1.2.1.12)], a glycolytic enzyme which is expressed constitutively in all cell types. Using defined segments of this cloned DNA as probes, we have determined the DNase I sensitive domain of the GAPDH natural gene in the hen oviduct. When nuclei isolated from hen oviduct were treated with DNase I under conditions known to preferentially degrade actively transcribed genes (i.e., 15-20% of the DNA rendered perchloric acid soluble), a region of approximately 12 kilobase(s) (kb) containing the GAPDH coding sequences and flanking DNA was found to be highly susceptible to digestion by DNase I. Approximately 4 kb downstream from the end of the coding sequences, there was an abrupt transition from the DNase I sensitive or "open" configuration to the resistant or "closed" configuration. The chromatin then remained in a closed conformation for at least 10 kb further downstream. On the 5' side of the gene, the transition from a sensitive to a resistant configuration was located about 4 kb upstream from the gene. In addition, we have localized two repeated sequences in the area of DNA that was cloned. One of these is of the CR1 family of middle repetitive elements. It is located about 18 kb 3' to the gene and as such lies well outside of the DNase I sensitive region which encompasses GAPDH. The other repetitive element is of an uncharacterized family. It is located upstream from the gene and appears to be within a region of transition from the DNase I sensitive to resistant states.     

DNA polymerases of ascites hepatoma cells. I. Purification and properties of a DNA polymerase from soluble fraction

DNA polymerase from soluble fraction of ascites hepatoma cells has been purified about 490-fold. The polymerase requires template DNA, all four deoxyribonucleoside triphosphates, and magnesium ions for the reaction. Optimal activity was found at pH 7.0 – 7.5, with 3 – 8 mM magnesium chloride, and 20 – 40 mM potassium phosphate. The purified enzyme utilizes preferentially DNA treated with pancreatic DNase as template.     

Nucleases in chloroplast of barley

Two barley chloroplast nuclease fractions were separated by the affinity chromatography and gel electrophoresis. Both were about 2 times more active to RNA than to native DNA and about half as active to denaturated DNA as to native DNA. Both fractions were as active to UV-irradiated (270 J m^-2) native DNA as to intact DNA but their action was inhibited by apurinic sites. The enzyme activities were inhibited by high concentrations of EDTA, NaCl, Mn²⁺, Ca²⁺, Zn²⁺ ions and by N-ethylmaleimide. They do not require Mg²⁺ ions but are stimulated or at higher concentration inhibited by their presence. Both RNase and DNase were active over a wide pH range (5.5–9), the optimum for DNase action in the presence of Mg²⁺ being 6.5, for RNA decomposing activity at pH 8.0. As no mononucleotides were detected in acid soluble form, it seems likely that DNase acts in the endonucleolytic way.     

Pea chloroplast DNA primase: characterization and role in initiation of replication

A DNA primase activity was isolated from pea chloroplasts and examined for its role in replication. The DNA primase activity was separated from the majority of the chloroplast RNA polymerase activity by linear salt gradient elution from a DEAE-cellulose column, and the two enzyme activities were separately purified through heparin-Sepharose columns. The primase activity was not inhibited by tagetitoxin, a specific inhibitor of chloroplast RNA polymerase, or by polyclonal antibodies prepared against purified pea chloroplast RNA polymerase, while the RNA polymerase activity was inhibited completely by either tagetitoxin or the polyclonal antibodies. The DNA primase activity was capable of priming DNA replication on single-stranded templates including poly(dT), poly(dC), M13mp19, and M13mp19_+ 2.1, which contains the AT-rich pea chloroplast origin of replication. The RNA polymerase fraction was incapable of supporting incorporation of ³H-TTP in in vitro replication reactions using any of these single-stranded DNA templates. Glycerol gradient analysis indicated that the pea chloroplast DNA primase (115–120 kDa) separated from the pea chloroplast DNA polymerase (90 kDa), but is much smaller than chloroplast RNA polymerase. Because of these differences in size, template specificity, sensitivity to inhibitors, and elution characteristics, it is clear that the pea chloroplast DNA primase is an distinct enzyme form RNA polymerase. In vitro replication activity using the DNA primase fraction required all four rNTPs for optimum activity. The chloroplast DNA primase was capable of priming DNA replication activity on any single-stranded M13 template, but shows a strong preference for M13mp19+2.1. Primers synthesized using M13mp19+2.1 are resistant to DNase I, and range in size from 4 to about 60 nucleotides.     

Biogenesis and Proteolytic Processing of Lysosomal DNase II

Deoxyribonuclease II (DNase II) is a key enzyme in the phagocytic digestion of DNA from apoptotic nuclei. To understand the molecular properties of DNase II, particularly the processing, we prepared a polyclonal antibody against carboxyl-terminal sequences of mouse DNase II. In the present study, partial purification of DNase II using Con A Sepharose enabled the detection of endogenous DNase II by Western blotting. It was interesting that two forms of endogenous DNase II were detected – a 30 kDa form and a 23 kDa form. Neither of those forms carried the expected molecular weight of 45 kDa. Subcellular fractionation showed that the 23 kDa and 30 kDa proteins were localized in lysosomes. The processing of DNase II in vivo was also greatly altered in the liver of mice lacking cathepsin L. DNase II that was extracellularly secreted from cells overexpressing DNase II was detected as a pro-form, which was activated under acidic conditions. These results indicate that DNase II is processed and activated in lysosomes, while cathepsin L is involved in the processing of the enzyme.     

Characterization of a novel insect digestive DNase with a highly alkaline pH optimum.

A novel DNase from the digestive tract of the spruce budworm (Choristoneura fumiferana) has been isolated and characterized. This DNase has two features that distinguish it from other known DNases: (1) it has a pH optimum of 10.5 to 11; (2) it plays an important role in the conversion of the insecticidal crystal protein from Bacillus thuringiensis to the active DNA-free toxin in the larval gut. Only one digestive DNase with an apparent molecular mass of 23 kDa was found and no associated carbohydrate was detected. It has some similarities to pancreatic DNase I in that divalent alkaline metal ion is required for activity and it is inhibited by monovalent cations. In particular, Mg(2+) and Ca(2+) were the most effective activators. Transition metal ions also activated the enzyme but were less effective. The enzyme is an endonuclease that hydrolyzes single and double stranded DNA but shows a higher specificity for single stranded DNA. The purified enzyme acted synergistically with proteases on crystals from Bacillus thuringiensis to yield the DNA-free toxin. To our knowledge, this is the first characterization of DNase activity in insect larvae and provides strong evidence that a DNase is an integral component of the larval digestive system.     

Purification and Properties of the Geminivirus Euphorbia Mosaic Virus

Euphorbia mosaic virus was purified from infected plants of Nicotiana benthamiana. Highest concentrations of virus particles were found in infected plant tissue between 10–12 days after inoculation. The enzyme driselase assisted in purification of the virus particles from the infected tissue yielding about 600 μg/kg of plant material. Purified preparations showed a maximum absorption at 260–263 nm and the ratio of absorption at 260 and 280 nm was 1.4. The viral nucleic acid was digestedby DNase I and S₁ Nuclease but not RNase A. A single coat protein with a MW of 32,000 d and two DNA bands with a MW 0.96 × 10⁶ d (2870 nucleotides) and 0.90 × 10⁶ d (2700 nucleotides) were associated with the purified virus particles. Virus specific DNA was isolated from infected tissue between 7 and 15 days after inoculations.     

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.