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:3.1.30.2 (endonuclease)
18,621 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Among 120 strains of gliding bacteria which were screened for restriction endonucleases, 27 were found positive. Additionally, three strains carried enzymes able to release the supercoiled state of closed circular DNA. By using a new rapid method, restriction endonuclease activity was released by stirring about 0.5 g of cells (fresh weight) in a motor-driven glass homogenizer in buffer containing Triton X-101, ethylenediaminetetraacetic acid, and mercaptoethanol. A yield from 60 to 80% of the total activity present in the cells was obtained with minimal destruction of the cells. The enzyme activity in the crude extract was measured semi-quantitatively by digestion of DNA and subsequent separation of the fragments on an agarose slab gel. The method appears to be generally applicable for the extraction of restriction endonucleases from gram-negative bacteria on an analytical scale and in a modified form for large-scale preparation of restriction enzymes.
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PMID:Restriction endonucleases: general survey procedure and survey of gliding bacteria. 10 30

An endonuclease (EtdBr DNase), which is more active in the presence of EtdBr, has been purified from yeast mitochondrial membrane (Jacquemin-Sablon, H., et al. (1979) Biochemistry 18 (preceding paper in this issue)). This paper deals with the analysis of the mechanism of this activation. Determination of the enzyme activity in the presence of intercalating and nonintercalating agents showed that the enzyme does not recognize the DNA structure modifiction provoked by drug intercalation. Studies carried out with a series of phenanthridinium derivatives led to the following model. The EtdBr DNase activation would result from the formation of a ternary complex, DNA--drug--Triton X-100. The activation capacity of a drug depends on its ability to bind simultaneoulsy to the DNA (not necessarily by intercalation) and the detergent. When this complex is formed, the DNA molecule is surrounded with Triton X-100 molecules which constitute an hydrophobic environment and make the substrate more prone to interaction with the enzyme. The implications of this model are discussed.
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PMID:Yeast mitochondrial deoxyribonuclease stimulated by ethidium bromide. 2. Mechanism of enzyme activation. 36 93

We have studied the properties of the high-Mr DNAse degrading single-stranded DNA which is present in extracts of Bacillus subtilis. This enzyme is a heterogeneous aggregate of identical subunits with an Mr of 36 000, as measured in dodecylsulfate/polyacrylamide electrophoresis. The aggregate can be disassembled by the presence of Triton X-100, but reforms spontaneously following removal of the detergent. A mild proteolytic treatment of the aggregate causes the irreversible and nearly quantitative conversion into the free subunit. The modified subunit has identical properties (in terms of size, chromatographic adsorption and catalytic activity) as the small DNAse previously described by Ciarrocchi et al. [Eur. J. Biochem. 61, 487 (1976)], i.e. an endonuclease highly specific for single-stranded DNA and producing 5'-P and 3'-OH ends.
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PMID:Properties of the high-molecular-weight deoxyribonuclease of Bacillus subtilis degrading single-stranded DNA. 41 55

tRNA-splicing endonuclease from the yeast Saccharomyces cerevisiae was purified to homogeneity greater than 5000-fold over a crude Triton X-100 extract of yeast total membranes, with 5% overall yield. This nuclear enzyme has the unusual heterotrimeric subunit structure alpha beta gamma (alpha = 31 kDa, beta = 42 kDa, and gamma = 51 kDa), as determined by sodium dodecyl sulfate gel electrophoresis, and has a molecular mass close to the sum of the three subunits, as determined by gel filtration of the native enzyme. From the purification, we estimate that there are approximately 100 molecules of endonuclease/cell.
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PMID:Yeast tRNA-splicing endonuclease is a heterotrimeric enzyme. 221 94

A stable DNA polymerase (EC 2.7.7.7) has been purified from the extremely thermophilic eubacterium Thermotoga sp. strain FjSS3-B.1 by a five-step purification procedure. First, the crude extract was treated with polyethylenimine to precipitate nucleic acids. The endonuclease activity coprecipitated. DEAE-Sepharose, CM-Sephrarose, and hydroxylapatite column chromatography were used to purify the preparation. As a final step on a small scale, preparative sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis was used. The purified DNA polymerase exhibited a molecular weight of 85,000, as determined by both SDS-polyacrylamide gel electrophoresis and size-exclusion chromatography. Its pH optimum was in the range pH 7.5-8. When assayed over the temperature range 30-80 degrees C, the maximum activity in a 30-min assay was at 80 degrees C. The enzyme was moderately thermostable and exhibited half-lives of 3 min at 95 degrees C and 60 min at 50 degrees C in the absence of substrate. Several additives such as Triton X-100 enhanced thermostability. During storage at 4 degrees C and -70 degrees C, the stability of the enzyme was improved by the addition of gelatin.
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PMID:Purification and some properties of a thermostable DNA polymerase from a Thermotoga species. 227 6

Endonuclease activity which specifically cleaves baseless (apurinic/apyrimidinic (AP] sites in supercoiled DNA has been purified from mitochondria of the mouse plasmacytoma cell line, MPC-11. Two variant forms separate upon purification; these have small but reproducible differences in catalytic and chromatographic properties, but similar physical properties. Both have a sedimentation coefficient of 4.0, corresponding to a molecular weight of 61,000 (assuming a globular configuration) and a peptide molecular weight of about 65,000 as determined by immunoblot analysis with antiserum raised against the major AP endonuclease from HeLa cells. Thus mitochondrial AP endonuclease appears to be a monomer of about 65 kDa, making it distinguishable from the major AP endonuclease of MPC-11 cells which, like those of other mammalian cells, appears to be a monomer of about 41 kDa. A possible 82-kDa precursor form was also detected by immunoblot analysis of a crude mitochondrial fraction. Mitochondrial AP endonuclease activity is greatly stimulated by divalent cations, has a pH optimum between 6.5 and 8.5, and cleaves the AP site by a class II mechanism to generate a 3'-OH nucleotide residue. These properties resemble those of the major mammalian AP endonucleases but, unlike those enzymes, mitochondrial AP endonuclease activity is neither inhibited by adenine or NAD+ nor stimulated by Triton X-100. Since the mitochondrial activity generates active primer termini for DNA synthesis, it could function in base excision DNA repair; alternatively, it might have a role in eliminating damaged mitochondrial genomes from the gene pool.
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PMID:Mitochondrial endonuclease activities specific for apurinic/apyrimidinic sites in DNA from mouse cells. 245 85

Three endonucleases from murine plasmacytoma cells that specifically nick DNA which was heavily irradiated with ultraviolet (UV) light were resolved by Sephacryl S-200 column chromatography. Two of these, UV endonucleases I and II, were purified extensively. UV endonuclease I appears to be a monomeric protein with a molecular mass of 43 kDa; UV endonuclease II has an S value of 2.9 S, with a corresponding molecular mass estimated at 28 kDa. Both enzymes act as a class I AP endonuclease, cleaving phosphodiester bonds via a beta-elimination mechanism, so as to form an unsaturated deoxyribose at the 3' terminus. Both have thymine glycol DNA glycosylase activity and their substrate specificities generally appear to be overlapping but not identical. UV endonuclease I acts on both supercoiled and relaxed DNAs, whereas II acts only on supercoiled DNA. Both enzymes are active in EDTA, but have different optima for salt, pH, and Triton X-100. Each enzyme is also present in cultured diploid human fibroblasts.
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PMID:Purification and characterization of UV endonucleases I and II from murine plasmacytoma cells. 246 92

A simple technique is proposed for detection of bacterial restriction endonucleases. Analysis is performed directly in the cells from colonies cultivated on Petri dishes. The cells collected with an inoculation loop are treated with lysozyme and Triton X-100. After centrifugation the supernatant is tested for endonuclease activity. The technique enables up to 100 colonies to be tested for 3-4 h.
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PMID:[A method for detecting restriction endonucleases in bacterial colonies]. 283 59

We describe an enzymatic procedure for exposure of single-stranded DNA (ssDNA) containing the halogenated pyrimidines (HdUrd) bromodeoxyuridine (BrdUrd) or iododeoxyuridine (IdUrd) in single cells to antibodies that bind to HrdUrd only in ssDNA. Production of ssDNA was accomplished by digesting the DNA using either restriction endonucleases alone or endonucleases followed by exonuclease III. The enzymatic production of ssDNA was maximal when 0.1 N HCl or 0.1 M citric acid plus Triton X-100 was added to extract nuclear proteins prior to enzymatic denaturation. The restriction endonucleases Bam HI, Dde I, Eco RI, and Hind III produced significant ssDNA when used alone to allow binding of detectable amounts of the anti-HdUrd antibody IU-4 in Chinese hamster ovary cells labeled with 10 microM BrdUrd or 10 microM IdUrd. However, these treatments did not expose sufficient ssDNA to allow binding of IU-1, an anti-HdUrd antibody with lower binding affinity. IU-4 binding was most intense after treatment with Eco RI. Treatment with exonuclease III following endonuclease digestion allowed substantially more IU-4 binding.
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PMID:Use of restriction endonucleases and exonuclease III to expose halogenated pyrimidines for immunochemical staining. 285 Jan 42

The catalytic properties of the HhaII restriction endonuclease were studied using plasmid pSK11 DNA containing a single 5'-G-A-N-T-C HhaII cleavage site as substrate. Reactions were followed by two methods: 1) gel electrophoretic analysis of nicked circular and linear DNA products, or 2) release of 32P-labeled inorganic phosphate from specifically labeled HhaII sites in a reaction coupled with bacterial alkaline phosphatase. The enzyme is optimally active at 37 degrees C in 10 mM Tris-HCl (pH 9.1) and 4-10 mM MgCl2 without added NaCl. Activity is stabilized by the presence of 2-mercaptoethanol and 0.2% Triton X-100 or 50 microgram/ml bovine serum albumin. At enzyme concentrations below 10 nM and using pSK11 as substrate, initial kinetic rates were dependent on the order of mixing of reactants. A lag of 3-4 min was observed if enzyme or substrate was added last. Preincubation of substrate and enzyme followed by initiation of the reaction with MgCl2 or preincubation of the enzyme with nonspecific DNA followed by initiation with substrate eliminated or reduced the lag, respectively, and speeded up the reactions. Under a wide range of reaction conditions, nicked pSK11 DNA accumulated early, while linear molecules appeared later, suggesting that HhaII cleaves one strand at a time in separate binding events. The apparent Km for covalently closed pSK11 DNA molecules was approximately 17 nM, and the turnover number for the conversion of covalent to nicked sites was 1.1 single strand scissions/min. Pre-steady state kinetic analysis indicated that cleavage of the first phosphodiester bond in a site is first order with a rate constant of about 0.8 min-1, while cleavage of the second phosphodiester bond is first order with a rate constant of about 0.2 min-1.
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PMID:Catalytic properties of the HhaII restriction endonuclease. 299 12


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