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)

Deoxyribonucleic acid polymerase-beta (EC 2.7.7.7) has been purified over 100 000-fold from a whole cell extract of guinea pig liver. The enzyme yields a single stainable band when subjected to non-denaturing polyacrylamide gel electrophoresis, and this band corresponds to the DNA polymerase activity when a sister gel is sliced and assayed. The final fraction has a specific activity of 21 000 units/mg; this value can be increased significantly by addition of various components, including glycols, polyamines or any of several protein factors which can be purified from the crude extract. The DNA polymerase-beta lacks detectable exonuclease or endonuclease activity, has an alkaline pH optimum and has a requirement for all four deoxyribonucleoside triphosphates, a divalent cation and a primer-template for maximal activity. While activated DNA is the preferred primer-template, the enzyme is capable of utilizing native and denatured DNA as well as several synthetic polynucleotides as primer-templates. The latter are especially effective when manganese is the divalent cation. Magnesium, at 10 mM, is the preferred divalent cation when activated DNA is used. Manganese, and to a lesser extent cobalt, can substitute for magnesium while zinc and calcium cannot. The beta-polymerase has a half-life of 10 min at 40 degrees C and this is increased in the presence of either DNA or NaCl. The enzyme is stimulated by glycols, polyamines and NaCal or KCl, and is inhibited by several known inhibitors of DNA polymerase activity including o-phenanthroline, heparin, organic solvents and sulfhydryl blocking agents. Guinea pig liver DNA polymerase-beta is remarkably similar to the rat Novikoff hepatoma beta-polymerase with respect to its isoelectric point of 8.4 and its molecular weight of 32 000 as determined by sucrose gradient centrifugation under high or low salt conditions or sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This similarity is further extended to the removal, at the final step in purification, of a protein capable of stimulating the homogeneous enzyme. Removal of this protein could explain the lower molecular weight of the guinea pig and other rodent-derived beta-polymerases, when compared to the beta-polymerases from other systems.
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PMID:Purification and properties of DNA polymerase-beta from guinea pig liver. 70 39

A type II restriction endonuclease (endo R . Bsp) has been purified from Bacillus sphaericus to electrophoretic homogeneity. The enzyme appears to be a single polypeptide chain with a molecular weight of 35000. Its pH optimum is around 8.2, it requires 20 mM Mg2+ for optimal activity and it is inhibited by Zn2+. The yield of the enzyme is higher than that of any type II restriction endonuclease so far reported. The enzyme also cleaves single-stranded DNA, albeit at a slower rate. It seems likely that single-stranded DNA is cleaved at the same sequences as double-stranded DNA. Bacillus sphaericus also contains a modification methylase (meth M . Bsp) which completely protects the cell's own DNA against cleavage by its restriction endonuclease. The methylase activity has been partially purified, it copurifies with the nuclease until the next to the last step. The enzyme does not require ATP or Mg2+, it transfers the methyl group of S-adenosyl-methionine to cytosine residues of DNA. As the action of this methylase completely protects any DNA from endo R . Bsp cleavage, it seems likely that the methylase recognizes and methylates the same sequence (dG-dG-dC-dC) as the nuclease.
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PMID:Biochemical characterization of the restriction-modification system of Bacillus sphaericus. 71 Apr 8

An endonuclease which is active with regard to depurinated, alkylated, arylated, and arylamidated DNA has been purified 500-fold from Micrococcus luteus. In this purification, separation from the pyrimidine-dimer-specific ultraviolet-endonuclease has been achieved. The enzyme has a molecular weight of 30000 on the basis of gel filtration; its activity is not absolutely dependent upon the presence of Mg2+, but 5--30 mM Mg2+ produces a five-fold stimulation. Potassium chloride concentrations of less than 100 mM are optimal, while concentrations exceeding 100 mM inhibit. The enzyme has no effect on native DNA, but introduces single-strand breaks into DNA containing apurinic/apyrimidinic sites produced by heating at an acidic pH. DNA treated with such carcinogens as N-alkyl-N-nitrosoureas, alkyl methanesulfonates, alkyl sulfates, nitrogen mustard, beta-propiolactone, 7-bromomethyl-benz[a]anthracene, N-acetoxy-2-acetylaminofluorene, and 7,12-dimethyl-benz[a]anthracene-5,6-oxide also becomes susceptible to enzymic action. The activity of the enzyme has been detected by making use of the difference in mobility between supercoiled closed-circular DNA of Pseudomonas phage PM2 and its nicked form in agarose gel elctrophoresis. Even depurinated or carcinogen-modified supercoiled PM2 DNA migrated faster than the respective relaxed nicked forms. A comparison of the number of enzyme-catalyzed single-strand breaks with the number of alkali-labile (i.e. apurinic) sites in carcinogen-modified PM2 DNA showed that the enzyme preparation introduced approximately twice as many breaks into the substrates as the number of apurinic sites present. We conclude that the enzyme preparation either recognizes both apurinic sites and DNA bases carrying carcinogenic residues or contains DNA glycosidase activity in addition to the endonuclease activity. Exposure of ultraviolet-irradiated PM2 DNA to the endonuclease preparation showed that pyrimidine dimers were not substrates. The yield of enzyme-catalyzed single-strand breaks found in ultraviolet-irradiated DNA was five times the number of alkali-labile sites present suggesting that minor photoproducts, possibly 5,6-saturated pyrimidine residues, were recognized in addition to apurinic sites.
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PMID:Purification and characterization of an endonuclease from Micrococcus luteus that acts on depurinated and carcinogen-modified DNA. 71 Apr 10

DNA-dependent RNA polymerase class C (or III) has been solubilized from either uninfected or adenovirus-2-infected HeLa cells and purified by chromatography on phosphocellulose, DNA-cellulose, CM-Sephadex and DEAE-Sephadex. The last column separated the enzyme into three forms CI, CII and CIII, which were completely free of RNA polymerases class A and B and of DNase and RNase. The total and the relative amount of these different enzyme C forms did not vary whether purified from uninfected or infected cells. Irrespective of the stage of purification, the three enzyme forms transcribed deproteinized adenovirus-2DNA very efficiently. This transcription was highly sensitive to elevated ionic strength (especially in the presence of Mg2+) and was accompanied by continuous reinitiation as shown by adding poly(rI), a potent inhibitor of initiation. In addition heparin-resistant initiation complexes could be formed at elevated temperature. The RNA synthesized in vitro on deproteinized intact adenovirus-2 DNA by the different forms of RNA polymerase class C, has been characterized. Analysis of the transcripts by gel electrophoresis, RNA self-annealing, hybridization to separated adenovirus-2 DNA strands and to restriction endonuclease (BamHI, HindIII), adenovirus-2 DNA fragments have demonstrated that restriction endonuclease (BamHI, HindIII), adenovirus-2 DNA fragments have demonstrated that the various regions of the adenovirus-2 genome were randomly transcribed. In addition, hybridization of RNA transcripts labelled at their 5' end by either [gamma32P]ATP or [gamma-32P]GTP indicated that not only elongation but also initiation occurred randomly through the entire adenovirus-2 genome, irrespective of the form of the enzyme and of the origin of the cells (normal or infected). The results are discussed in terms of the components which are possibly involved in specific transcription.
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PMID:Transcription in vitro of adenovirus-2 DNA by RNA polymerases class C purified from uninfected and adenovirus-infected HeLa cells. 71 Apr 51

The specific endonuclease Bam HI from Bacillus amyloliquefaciens (RUB 500) has been purified to apparent homogeneity. Two active forms of the enzyme corresponding to the dimeric and tetrameric forms have been isolated. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the enzyme dissociated into Mr = 22,000 +/- 500 subunits. Bam HI has a broad pH optimum on the alkaline side and requires Mg2+ which can be partially replaced by Mn2+. The enzyme catalysis appears to be governed by a two-step mechanism.
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PMID:Purification and characterization of the sequence-specific endonuclease Bam HI. 76 9

The enzymes involved in host-controlled modification and restriction by Bacillus subtilis strain N were detected in cell free extracts. In the presenct of Mg2+ the N-specific endonucleases cleaved unmodified DNA but did not attack phi-105C. N DNA carrying N-specific modification. The restriction endonuclease required neither SAM nor ATP for its activity. The N-specific modification enzyme was active only in the presence of SAM, indicating that modification in this syteem is a methylation of DNA.
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PMID:In vitro modification and restriction of phage phi-105c DNA with Bacillus subtilis N cell-free extract. 80 94

Crude extracts of cultured human lymphoblasts (CCRF-CEM) contain endonuclease activity that cleaves ultraviolet-irradiated DNA in preference to untreated DNA. Purification of this activity was carried out using ultraviolet-irradiated PM2 phage DNA (5000 ergs/mm2) as substrate in the enzyme assay. Since endonuclease specific for depurinated or depyrimidinated DNA might account for the apparent ultraviolet-irradiated DNA-specific activity, fractions derived during purification were also assayed with partially depurinated DNA. Chromatography of a 45-60% (NH4)2SO4 fraction on a Sephadex G-100 column yielded a peak of activity (35 000 daltons) highly active against depurinated DNA but also active for ultraviolet-irradiated DNA. Further purification by DEAE-cellulose chromatography resolved two activities. One was highly specific for depurinated DNA with only minor activity for ultraviolet-irradiated DNA, and was strongly stimulated by Mg2+. The other was non-specifically active against ultraviolet-irradiated or untreated DNA and was independent of Mg2+. Additional studies suggest that neither of these two activities but a third enzyme is responsible for the ultraviolet-irradiated DNA-specific endonuclease activity observed in crude cell extracts.
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PMID:Partial purification of endonuclease activity from human lymphoblasts. Separation of activities for depurinated DNA and DNA irradiated with ultraviolet light. 81 Jan 75

An endonuclease present in partially purified preparations of calf thymus DNA polymerase has been purified to homogeneity. It has a molecular weight of 53,000 +/- 2,500 as determined by sucrose gradient sedimentation. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate indicates the protein is composed of four subunits, each polypeptide possessing a molecular weight of 13,000. Its isoelectric point is 10.3 +/- 0.2. The endonuclease has a pH optimum at 6.6, requires Mg2+ or Mn2+ for activity, and does not attack RNA. The enzyme appears to be present in tissues other than calf thymus. The enzyme catalyzes the endonucleolytic cleavage of both denatured and native eukaryotic DNA. The enzyme introduces a limited number of single strand nicks into native DNA; hydrolysis of denatured DNA produces acid-soluble oligonucleotides. The average size of the limit product, sedimented in an alkaline sucrose gradient, is 1200 nucleotides for native DNA. The product contains 5'-phosphoryl and 3'-hydroxyl termini. While all four deoxynucleotides are found at the 5' termini, pyrimidine residues predominate. Calf thymus DNase V degrades closed circular duplex SV40 DNA and glucosylated T4DNA but not poly(dA-dT). The rate of hydrolysis of homopolymers is: poly(dT) greater than poly(dA) greater than poly(dC) greater than poly(dG) in the presence of Mg2+, and poly(dT) greater than poly(dC) greater than poly(dA) = poly(dG) in the presence of Mn2+.
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PMID:Mammalian endonuclease, DNase V. Purification and properties of enzyme of calf thymus. 83 11

An endonuclease specific for apurinic sites in double-stranded DNA has been partially purified from calf liver extracts. The enzyme has a pH optimum of 9.5, is only slightly stimulated by low concentrations of Mg2+, and has a molecular weight of 28 000. Inhibitors of the endonuclease include Ca2+, EDTA, p-HOHgBzO, NaCl, and tRNA. The enzyme introduces single- and double-stranded breaks in depurinated DNA. High concentrations of the enzyme preparation degrade untreated single-stranded DNA, but not ultraviolet (UV) irradiated DNA or DNA treated with methylmethanesulfonate or 7-bromomethyl-12-methylbenz[a]-anthracene. Enzymatic incisions produce 3'-hydroxyl and 5'-phosphate end groups. Some of the properties of the calf liver apurinic endonuclease differ from those of a similar endonuclease obtained from calf thymus by S. Ljungquist and T. Lindahl [(1974), J. Biol. Chem. 249, 1530] and in this laboratory. The data suggest that these are isozymes.
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PMID:An endonuclease from calf liver specific for apurinic sites in DNA. 84 21

1. An endonuclease activity from a cultured human lymphoblast cell line, CCRF-CEM, was further purified by chromatography on phosphocellulose to remove a nonspecific acid endonuclease. 2. The purified enzyme acted quantitatively on apurinic sites in the DNA of PM2 phage. It showed optimum activity over a broad range of pH (7.0--8.5), was absolutely dependent on Mg2+ (optimum concentration 0.5 mM) and did not have detectable activity against intact DNA. 3. This enzyme was used as a probe to estimate the number of apurinic or apyrimidinic lesions induced in PM2 DNA by either ultraviolet or X-ray irradiation. High doses of ultraviolet radiation (2500 to 5000 J/m2) immediately induced 0.2 to 0.4 endonuclease-susceptible lesions per molecule of DNA. The lesions continued to increase for several hours after irradiation, reaching a level more than double that found initially. By contrast, in DNA exposed to 5000 rads of X-ray irradiation, the number of endonuclease-susceptible sites reached a maximum of about 0.6 per molecule immediately after exposure and did not increase further. It thus appears that ultraviolet-irradiated (but not X-ray irradiated) DNA contains damaged bases that are lost spontaneously after irradiation. 4. A second endonuclease was purified and was shown to act on ultraviolet-induced lesions that are distinct from either apurinic or apyrimidinic sites. These new lesions occur about ten times more frequently than ultraviolet-induced apurinic or apyrimidinic sites.
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PMID:Purification and characterization of human endonucleases specific for damaged DNA. Analysis of lesions induced by ultraviolet or x-radiation. 99 Mar 19


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