Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.25.1 (deoxyribonuclease)
1,471 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The presence of a nuclear DNA polymerase in mouse sperm from adult testes has been confirmed and the properties of this enzyme further investigated. This activity was shown to be greatly enhanced by treating the spermatozoa with methanol or ethanol before incubation in the reaction medium or by their addition in small amounts to this medium. It was protected against degradation by nuclear proteases by adding soybean trypsin inhibitor and was stimulated by ATP. It was found to be Mg2+ dependent (optimum concentration: 7.5 mM), DNA dependent, and all four deoxynucleoside triphosphates were needed for optimal reaction. The radioactive acid-precipitable product of polymerization was not eliminated by organic solvents, nor by pronase, ribonuclease or by nuclease S1; however, it was converted to a large extent to acid-soluble products by pancreatic deoxyribonuclease. Since it was only partially solubilized by Triton X-100, it therefore did not appear to be preferentially associated with the nuclear membranes. The activity recovered after incubation depended also on the pH (optimum at pH 8.3) and did not work well in a medium for DNA polymerase alpha. The temperature for maximum incorporation of nucleotides was found to be 32 degrees C and, under our conditions, the reaction was linear for 30 min. The DNA polymerase activity was inhibited by low and high concentrations of KCl. It was not lowered by N-ethylmaleimide or p-hydroxymercuribenzoate; urea slightly stimulated the reaction and this stimulation was reversed by subsequent treatment with N-ethylmaleimide. Actinomycin D (40 mug/ml), ethidium bromide (25--50 muM), netropsin (5--50 mug/ml), and spermidine (0.5--2.5 mM) lowered the polymerization of DNA precursors. The nuclear enzyme could shift from the endogenous template to activated exogenous calf thymus DNA, the resulting nuclear radioactivity being reduced. The endogenous DNP template ability was not increased by deoxyribonuclease activation according to the method of Aposhian and Kornberg (J. Biol. Chem. (1962) 237, 519--525) suggesting that the amount of DNA polymerase associated with chromatin was probably limiting the reaction. The DNA polymerase activity detected in mouse sperm nuclei has numerous properties of low molecular weight DNA polymerases (DNA polymerase beta) reported in several eukaryotic organisms.
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PMID:Further characterization of a DNA polymerase activity in mouse sperm nuclei. 1 3

DNA polymerase was purified from Drosophila melanogaster embryos by a combination of phosphocellulose adsorption, Sepharose 6B gel filtration, and DEAE-cellulose chromatography. Three enzyme forms, designated enzymes I, II, and III, were separated by differential elution from DEAE-cellulose and were further purified by glycerol gradient centrifugation. Purification was monitored with two synthetic primer-templates, poly(dA) . (dT)-16 and poly(rA) . (dT)-16. At the final step of purification, enzymes I, II, and III were purified approximately 1700-fold, 2000-fold and 1000-fold, respectively, on the basis of their activities with poly(dA) . (dT)-16. The DNA polymerase eluted heterogeneously as anomalously high-molecular-weight molecules from Sepharose 6B gel filtration columns. On DEAE-cellulose chromatography enzymes I and II eluted as distinct peaks and enzyme III eluted heterogeneously. On glycerol velocity gradients enzyme I sedimented at 5.5-7.3 S, enzyme II sedimented at 7.3-8.3 S, and enzyme III sedimented at 7.3-9.0 S. All enzymes were active with both synthetic primer-templates, except the 9.0 S component of enzyme III, which was inactive with poly(rA) . (dT)-16. Non-denaturing polyacrylamide gel electrophoresis did not separate poly(dA) . (dT)-16 activity from poly(rA) . (dT)-16 activity. The DNA polymerase preferred poly(dA) . (dT)-16 (with Mg2+) as a primer-template, although it was also active with poly(rA) . (dT)-16 (with Mn2+), and it preferred activated calf thymus DNA to native or heat-denatured calf thymus DNA. All three primer-template activities were inhibited by N-ethylmaleimide. Enzyme activity with activated DNA and poly(dA) . (dT)-16 was inhibited by K+ and activity with poly(rA) . (dT)-16 was stimulated by K+ and by spermidine. The optimum pH for enzyme activity with the synthetic primer-templates was 8.5. The DNA polymerases did not exhibit deoxyribonuclease or ATPase activities. The results of this study suggest that the forms of DNA polymerase from Drosophila embryos have physical properties similar to those of DNA polymerase-alpha and enzymatic properties similar to those of all three vertebrate DNA polymerases.
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PMID:Three forms of DNA polymerase from Drosophila melanogaster embryos. Purification and properties. 9 4

Protease-free bovine pancreatic deoxyribonuclease (DNase) (1.6 X 10(-4) mmol) was thiolated on the NH2 groups with N-acetyl-DL-homocysteine thiolactone (2.4 X 10(-2) mmol) at pH 10.5 with imidazole (2.4 X 10(-2) mmol) as the catalyst in the presence of 4,4'-dithiodipyridine (4.2 X 10(-2) mmol). The product obtained after 16 h at 4 degrees C, 2-acetamido-4-(4'-dithiopyridyl)butyryl-DNase, isolated by gel filtration, contained an average of 0.87 +/- 0.13 mol of mixed disulfide per mol of DNase. Ribonuclease (RNase) was thiolated in a similar manner, but under N2 in the absence of 4,4'-dithiodipyridine. The protein N-acetylhomocysteinyl-RNase contained on the average 0.94 +/- 0.11 mol of sulfhydryl groups per mol of RNase. The coupling of RNase ot DNase was accomplished by thiol-disulfide interchange at pH 6.2 and 25 degrees C for 90 min. The hybrid enzyme (yield 25--33%, based upon the DNase derivative used) was freed from unreacted DNase, RNase, and homodimers by gel filtration, affinity chromatography, and salting-out chromatography. The purified enzyme contained one molecule each of DNase and RNase and hydrolyzed thymus deoxyribonucleic acid (DNA) and yeast or transfer ribonucleic acid (RNA) with 75 and 40% of the efficiencies, respectively, of the parent enzymes. The RNA strand of the hybrid substrate, phage f1 DNA-[3H]RNA, prepared from phage DNA with RNA polymerase, was hydrolyzed rapidly by the hybrid enzyme but was not hydrolyzed by RNase alone. A conjugate of the two enzymes offers the possibility in vivo of delivering two enzymes that differ in size, charge, and biological function to the same site at the same time.
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PMID:Preparation of the bifunctional enzyme ribonuclease-deoxyribonuclease by cross-linkage. 48 31

A ribonuclease-sensitive DNA polymerase, which uses an endogenous template, is detectable in the 39,000 g supernatant of a rat thymus homogenate, and appears as a single peak of activity in the void volume after Sephadex G 150 or G 200 gel filtration chromatography. Native and "activated" DNA-dependent DNA polymerase activities coincide with the endogenous-templated polymerase activity. Treatment of the thymus extract with ribonuclease(s) prior to gel filtration chromatography yields two other peaks of activity in addition to the void volume peak. The appearance of the two lower molecular weight peaks of activity is accompanied by a concomitant decrease in the endogenous-templated activity. The effect of ribonuclease is specific and cannot be reproduced by a similar deoxyribonuclease treatment.
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PMID:DNA polymerase activity associated with endogenous template: release by ribonuclease treatment. 80 37

The recently isolated neutral deoxyribonuclease from crab (Cancer pagurus) testes has been characterized in its mode of action and its specificity. The enzyme is a typical endonuclease, forming 5'-phosphate oligonucleotides of large average size; after extensive digestion of calf thymus DNA over 75% of the fragments have a size larger than pentanucleotides and mononucleotides are absent. As far as specificity is concerned, thymidine is very abundant in the 5'-penultimate position (approximately 50%) and in the 3'-terminal position (37%) and almost absent in the 5'-terminal position (approximately 1%), the values quoted concerning Escherichia coli digests of average size (Pn) between 50 and 10.
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PMID:The specificity of a neutral deoxyribonuclease from Cancer pagurus. 123 41

The location of the phosphodiester bond cleaved by homogeneous Mg2+-dependent apurinic endodeoxyribonuclease (EC 3.1.25.2; APE) of bovine calf thymus has been determined by using a 21-mer oligonucleotide containing a single central apurinic site as a substrate. A single product of cleavage consistent with cleavage of the oligonucleotide 5' to the apurinic site, and leaving a 3' hydroxyl group, was identified. This enzyme is, therefore, a class II apurinic endonuclease. The substrate specificities of this enzyme have been determined by using a variety of natural and synthetic DNAs or oligonucleotides containing base-free sites. Calf thymus APE has an absolute requirement for a double-stranded DNA and requires an abasic site as a substrate. The presence of a base fragment such as a urea residue, an alkoxyamine group attached to the C'-1 position of the abasic site, or reduction of the C'-1 aldehyde abolishes the APE activity of this enzyme. Synthetic abasic sites containing either ethylene glycol, propanediol, or tetrahydrofuran interphosphate linkages are excellent substrates for bovine APE. These results indicate that APE has no absolute requirement for either ring-opened or ring-closed deoxyribose moieties in its recognition of DNA-cleavage substrates. The enzyme may interact with the pocket in duplex DNA that results from the base loss or with the altered conformations of the phosphodiester backbone that result from the abasic site.
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PMID:Mechanism of DNA cleavage and substrate recognition by a bovine apurinic endonuclease. 247 77

This paper describes the use of methoxyamine to study the enzymatic reactions catalyzed by uracil-DNA glycosylase and by AP (apurinic/apyrimidinic) endodeoxyribonuclease isolated from mammalian cells. [14C]Methoxyamine permits one to follow the formation of AP sites in a uracil-containing polydeoxyribonucleotide incubated with calf thymus uracil-DNA glycosylase. The number of methoxyamine-reacted AP sites is equal to that of uracil released. Methoxyamine has no effect on the uracil-DNA glycosylase activity and may be added together with the enzyme in order to block the AP sites and prevent the degradation of the polynucleotide by the AP endonucleases that may be present in a crude preparation. Addition of methoxyamine to AP sites prevents not only the enzymatic hydrolysis of the adjacent phosphodiester bond but also the degradation of the polynucleotide by NaOH. This protective effect disappears after methoxyamine is removed by acetaldehyde.
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PMID:A new approach to the study of the base-excision repair pathway using methoxyamine. 258 Aug 33

Preparations of streptococcal DNAse D with high specific activity and free of other streptococcal nucleases have been obtained by zone electrophoresis and column chromatography. Antisera prepared by injecting rabbits with such preparations specifically neutralize the activity of this enzyme. As with DNAse B, preparations of DNAse D regularly exhibit ribonuclease activity. For both B and D enzymes, the order of substrate preference is thymus DNA, yeast RNA, bacterial RNA; but the specific activity of the D enzyme is higher than that of the B enzyme with respect to thymus DNA and lower with respect to bacterial RNA. Both the deoxyribonuclease and the ribonuclease activities exhibited by preparations of both enzymes are inhibited by bacterial RNA, but approximately 100-fold greater concentrations of bacterial RNA are required to achieve inhibition of the deoxyribonuclease activity of the D enzyme equivalent to the inhibition of the B enzyme. The deoxyribonuclease activity of the D enzyme is also inhibited by yeast RNA, but even larger amounts are required. These observations indicate that the D enzyme is immunologically distinct from the other streptococcal nucleases and that it differs quantitatively from the B enzyme with respect to relative specific activities on different substrates and behavior in the presence of the bacterial ribonucleic acid inhibitor.
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PMID:Streptococcal nucleases. II. Characterization of DNAse D. 496 68

Growth of L1210 leukemia cells which had been previously incubated with thymus DNA was inhibited. Leukemia-cell DNA did not affect tumor growth under similar conditions. Pretreatment of the thymus DNA with deoxyribonuclease suppressed the DNA induced inhibition. Both ribonucleasetreated DNA and untreated DNA inhibited tumor growth.
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PMID:Inhibition of L1210 tumor growth by thymus DNA. 582 48

The immune response of congenitally athymic (nude) mice induced by immune ribonucleic acid (iRNA) to lipopolysaccharides of Escherichia coli 0-55 (LPS) was studied. The thymus-independent nature of the immune response of mice to LPS was confirmed and nude mice responded to LPS in a manner similar to normal mice. An iRNA preparation extracted from the spleen of nude mice immunized with LPS could induce the proliferation of rosette-forming cells (RFC) in nude mice. iRNA preparations were insensitive to treatment with deoxyribonuclease and pronase, but were inactivated by ribonuclease treatment. The active fraction of the iRNA preparation had sedimentation values in a sucrose density gradient between 7 and 16 S and comprised only a small fraction of the total RNA present in the spleen cells, thereby indicating that the active moiety was one or more species of RNA. The anamnestic response was induced by treatment with iRNA made from the spleen of nude mice immunized with LPS. An increase in the number of rosette-forming cells (RFC), plaque forming cells (PFC) and formation of humoral antibody to LPS was seen after injection with a small amount of LPS 4 weeks after iRNA treatment.
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PMID:Cell preparation in immune response by immune ribonucleic acid. II. Independence of T lymphocytes in immune response against T-independent antigens. 616 81


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