Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.7.7 (DNA polymerase)
 17,007 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Enzymes of deoxyribonucleotide and DNA biosynthesis, which are little known in plants, were studied in root tips of germinating broad beans (Vicia faba) and in fast-growing cultures of soybean cells (Glycine max). The plant cells contain a ribonucleoside 5'-diphosphate reductase which is detected in vitro only during a limited period of growth, viz. 30--32 h after inhibition of Vicia seeds, and between the second and third day after inoculation of soybean cultures. In both species ribonucleotide reductase activity precedes maximum DNA synthesis. The reductases could be precipitated with ammonium sulfate but were not purified further due to the extremely low enzyme content of the plant extracts. Therefore the reductive pathway of deoxyribotide formation was also established in Vicia root tips by efficient labeling of the plant DNA with a ribonucleoside, [5-3H]cytidine, which reaches a maximum at the same time as the reductase activity measured in vitro. Cycloheximide inhibits this process, indicating the need for de novo enzyme induction. In contrast, DNA polymerase is present in the tissue throughout the entire development and rises only 2-fold in activity during the S phase. The soluble polymerases were partially characterized in both legume species and were found very similar to the DNA polymerase of pea seedlings. Ribonucleotide reductase is more likely a limiting component of DNA formation during the plant cell cycle than DNA polymerase.
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PMID:Deoxyribonucleotide synthesis and DNA polymerase activity in plant cells (Vicia faba and Glycine max). 42 Aug 54

Synthesis of phiX174 viral (+) strand circles in vitro requires gene A protein, rep protein, DNA binding protein, and DNA polymerase III holoenzyme (Eisenberg, S., Scott, J. F., and Kornberg, A., (1976) Proc. Natl. Acad. Sci. U.S.A. 73, 3151-3155). We have used this reaction as an assay to isolate gene A protein in greater than 90% purity. Its molecular weight under denaturing conditions is 59,000. The protein tends to aggregate and lose activity at low ionic strength. Tritium-labeled gene A protein cleaves the phiX174 duplex replicative form and is bound to it in a 1:1 ratio as part of an active replication complex. The attachment, at the 5' phosphoryl end of the cleavage point, is apparently covalent. The complex was not dissociated by: (i) banding in CsCl, (ii) treatment with 0.2 M NaOH, or (iii) boiling in 1% sodium dodecyl sulfate and electrophoresis on a sodium dodecyl sulfate-acrylamide gel; only micrococcal nuclease digestion of the DNA released the protein.
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PMID:Purification and characterization of phiX174 gene A protein. A multifunctional enzyme of duplex DNA replication. 44 53

The vaccinia virus-induced DNA polymerase has been purified about 500-fold from a cytoplasmic extract of vaccinia-infected HeLa cells. Analysis of the purified fraction by sodium dodecyl sulfate-polyacrylamide gel electrophoresis reveals a single polypeptide of 110,000 daltons, which is greater than 95% pure. This polypeptide co-sediments with polymerase activity through a glycerol gradient. The sedimentation coefficient of the enzyme is 6.3 S, and its Stokes radius is 4.6 nm. The molecular weight of the native enzyme derived from these values is 115,000. Vaccinia polymerase is therefore a single large polypeptide of 110,000 to 115,000 daltons. The purified fraction has no significant endonuclease activity, but a strong exonuclease activity co-purifies with polymerase activity through every step in the isolation. The polymerase and exonuclease activities are inactivated at 45 degrees C at the same rate. It is likely, therefore, that both activities are catalyzed by the same polypeptide. The exonuclease hydrolyzes DNA predominantly in the 3' leads to 5' direction, to produce 5' mononucleotides. The exonuclease degrades single-stranded DNA more rapidly than duplex DNA, and the rate of digestion of both single-stranded and double-stranded DNA increases as the size of the substrate decreases. Single-stranded circular DNA is a potent inhibitor of the exonuclease activity, but duplex circular DNA has no significant effect on its activity.
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PMID:Purification and properties of the deoxyribonucleic acid polymerase induced by vaccinia virus. 46 91

During development of the rat anterior pituitary gland (APG) there is a fall in DNA replication which is accompanied by a decline in the activity of the soluble DNA polymerase and of an endonuclease. This latter enzyme is capable of activating the DNA template for the DNA polymerase assay. Sulpiride sulfate, a drug known to produce prolactin release from the APG, increases thymidine incorporation in the APG 20 h after the injection. This drug also enhances the activity of the soluble DNA polymerase while that of the endonuclease and thymidine kinase does not change. The results suggest that the intracellular prolactin content regulates DNA replication in mammotrophs and that the soluble DNA polymerase plays an important role in this regulation.
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PMID:DNA synthesis in the pituitary gland of the rat. Effect of sulpiride and postnatal maturation. 47 Nov 96

We have purified the DNA polymerase from Mycoplasma orale to homogeneity. The protein structure of the enzyme was declined by sodium dodecyl sulfate gel electrophoresis, which revealed a single band of 116 000 daltons that was coincident with the polymerase activity profile in the final step of DNA--cellulose chromatography, and by two-dimensional gel analysis, which demonstrated a single protein species at pI = 6.8 that was congruent with enzyme activity and contained the same 116 000 polypeptide. although severe enzyme aggregation occurs during nondenaturing gel electrophoresis, a monomer species can be resolved with a Mr of 140 000 by the Ferguson plot analysis. Gel filtration and velocity gradient centrifugation yield a Stokes radius of 4.8 nm and a sedimentation coefficient of 5.6 S, respectively, from which Mr values of 106 000--128 000 can be computed. The different size values suggest that the polymerase molecule is asymmetric. The purified enzyme has a specific activity of approximately 6 x 10(5) units/mg of protein and in completely devoid of exodeoxyribonuclease and endodeoxyribonuclease activities, at exclusion limits of 10(-4)--10(-6%) of the polymerase activity. The mechanism of polymerization is moderately processive, with an average of 14 +/- 4 nucleotides incorporated per binding event, and the "effective template length" on activated DNA is approximately 40 nucleotides.
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PMID:Structural and enzymological characterization of the homogeneous deoxyribonucleic acid polymerase from Mycoplasma orale. 49 65

Terminal deoxynucleotidyltransferase is an enzyme which has been found to be associated with thymus cells, bone marrow cells, as well as leukocytes from patients with acute lymphoblastic leukemia and chronic myelocytic leukemia in blast crisis. We report here the purification of terminal deoxynucleotidyltransferase by an oligonucleotide affinity (oligo(dT)12-18 cellulose) column. By using a 35 to 70% (NH4)2SO4 cut, Sephacryl S200 column and an oligo(dT) cellulose column, terminal deoxynucleotidyltransferase has been purified from calf thymus cells to a specific activity of more than 8,500 units/mg of protein. The terminal deoxynucleotidyltransferase purified by this method contains no detectable DNA-dependent DNA polymerase or endonuclease activities. Furthermore, sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the enzyme appears to be homogeneous, with two polypeptides corresponding to the two subunits alpha (10,000) and beta (23,000) of terminal deoxynucleotidyltransferase. These data indicate that oligo(dT)12-18 cellulose can be used as a rapid and selective affinity column for the purification of terminal deoxynucleotidyltransferase.
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PMID:Purification of terminal deoxynucleotidyltransferase by oligonucleotide affinity chromatography. 64 3

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

The repeating sequence polymer d(A-I-C)n-d(I-C-T)n has been prepared using the chemically synthesized oligomers d(A-G-C)4 and d(C-T-G)4 and the DNA polymerase from Micrococcus luteus. The enzymatically synthesized polymer was used as template for preparation of d(A-G-C)n-d(G-C-T)n. Both deoxyribonucleotide polymers were characterized by nearest neighbor analyses, buoyant density measurements in cesium chloride and cesium sulfate, melting temperature, and circular dichroism (CD) spectra. The ribopolymers r(A-I-C)n-r(I-C-U)n and r(A-G-C)n-r(G-C-U)n were transcribed from d(A-I-C)n-d(I-C-T)n, and their CD spectra were compared with those of the respective deoxyribonucleotide polymers.
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PMID:Preparation and properties of the repeating sequence polymers d(A-I-C)n-d(I-C-T)n and d(A-G-C)n-d(G-C-T)n. 76 59

T7 DNA polymerase (DNA nucleotidyltransferase; deoxynucleosidetriphosphate:DNA deoxynucleotidyltransferase, EC 2.7.7.7) is composed of an 84,000 dalton protein specified by the gene 5 of the phage and a 12,000 dalton protein (TsnC protein) specified by the tsnC gene of E. coli [Modrich, P. & Richardson, C. C. (1975) J. Biol. Chem. 250 5515-5522]. Both proteins are necessary for T7 DNA polymerase activity and for the replication of T7 DNA. The TsnC protein is identical to thioredoxin of E. coli by the following criteria: (1) Homogeneous preparations of both proteins have TsnC and thioredoxin activity. (2) Both proteins show similar stability to heat. (3) They have identical mobilities, corresponding to a molecular weight of 12,000, on polyacrylamide gels containing sodium dodecyl sulfate. (4) Their amino-acid compositions are indistinguishabe. (5) Antibody prepared against thioredoxin inhibits TsnC activity. (6) TsnC protein isolated from purified T7 DNA polymerase has thioredoxin activity. In addition, preparations of T7 DNA polymerase itself exhibit thioredoxin activity and are partially inhibited by antibody to thioredoxin.
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PMID:Escherichia coli thioredoxin: a subunit of bacteriophage T7 DNA polymerase. 76 86

A simple and reproducible procedure is described which allows the fast and almost quantitative removal of DNA polymerases I and II from DNA polymerase III, in crude extracts of polA+ strains of Bacillus subtilis. The procedure entails streptomycin sulfate and ammonium sulfate fractionations; subsequent analysis of the partially purified preparation by G-200 chromatography, DEAE cellulose chromatography and density gradient sedimentation, shows that the ammonium sulfate fraction contains less than 5% of the total activity as DNA polymerase I and less than 2% as DNA polymerase II. The purification procedure, up to the ammonium sulfate step, was utilized for the analysis of the level of DNA polymerase III in several B. subtilis mutants, with results comparable to those obtained from the corresponding polA- strains following more cumbersome purification procedures. The M.W. of the purified form is of 227.000, somewhat greater than the published values. The early fractions of the purification have revealed the existence of a form with a M.W. of 426.000; the nature of this form, which has been observed in several instances and which is very unstable and short-lived, is under investigation.
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PMID:A simplified procedure for the analysis of DNA polymerase III levels in Bacillus subtilis strains. 82 85


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