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:2.7.7.7 (DNA polymerase)
17,007 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The effect of UV irradiation on the extent and fidelity of DNA synthesis in vitro was studied by using homopolymers and primed single-stranded varphiX174 phage DNA as substrates. Unfractionated and fractionated cell-free extracts from Escherichia coli pol(+) and polA1 mutants as well as purified DNA polymerase I were used as sources of enzymatic activity. (DNA polymerases, as used here, refer to deoxynucleosidetriphosphate:DNA deoxynucleotidyltransferase, EC 2.7.7.7.) The extent of inhibition of DNA synthesis on UV-irradiated varphiX174 DNA suggested that pyrimidine dimers act as an absolute block for chain elongation by DNA polymerases I and III. Experiments with an irradiated poly(dC) template failed to detect incorporation of noncomplementary bases due to pyrimidine dimers. A large increase in the turnover of nucleoside triphosphates to free monophosphates during synthesis by DNA polymerase I on irradiated varphiX174 DNA has been observed. We propose that this nucleotide turnover is due to idling by DNA polymerase (i.e., incorporation and subsequent excision of nucleotides opposite UV photolesions, by the 3'-->5' "proofreading" exonuclease) thus preventing replication past pyrimidine dimers and the potentially mutagenic event that should result. In support of this hypothesis, DNA synthesis by DNA polymerase from avian myeloblastosis virus and by mammalian DNA polymerase alpha, both of which are devoid of any exonuclease activity, was found to be only partially inhibited, but not blocked, by UV irradiation of the template and accompanied by an increased incorporation of noncomplementary nucleotides. It is suggested that UV mutagenesis in bacteria requires an induced modification of the cellular DNA replication machinery, possibly an inhibition of the 3'-->5' exonuclease activity associated with DNA polymerases.
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PMID:Mechanism of ultraviolet-induced mutagenesis: extent and fidelity of in vitro DNA synthesis on irradiated templates. 35 43

The replication of the Bacillus subtilis bacteriophages SPP-1 and phi 105 is sensitive to 6-(p-hydroxyphenylazo)-uracil (HPUra), a selective inhibitor of replicative DNA synthesis of B. subtilis which acts specifically at the levels of a replication-specific polymerase, DNA polymerase III (pol III). The origin of the HPUra-sensitive polymerase required for phage replication was examined by comparison of the drug sensitivity of phage development in a normosensitive host with that in a host carrying azp-12, a polC mutation that specifies production of an HPUra-resistant pol III. azp-12 specified HPUra-resistant phage host pol III. The host polIII requirement for SPP-1 replication also was confirmed by the demonstration that phage development was temperature sensitive in a host mutant carrying the polC mutation mut-1 (ts). Examination of the pol III activity of crude and purified cell-free preparations derived from phage-infected cells did not indicate any detectable changes in the specific activity, purification behavior, or drug sensitivity of the enzyme.
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PMID:Bacillus subtilis DNA polymerase III is required for the replication of DNA of bacteriophages SPP-1 and phi 105. 40 98

In vitro inhibitions by coumermycin A1 of DNA and RNA synthesis in toluenized cells were studied. In a sensitive strain, 50% inhibitions of replication and transcription were observed at 0.035 and 0.600 mug/ml, respectively. DNA synthesis in a toluenized-resistant mutant was 50% inhibited at 0.140 mug/ml of coumermycin A1, whereas RNA synthesis was unaffected at all concentrations tested. Studies with a mixture of toluenized-sensitive and -resistant bacteria ruled out the presence of a diffusable activator or inhibitor of coumermycin A1 action. Density label studies with toluenized pol A+ and pol A- strains indicated that replicative DNA synthesis was specifically inhibited, in agreement with the in vivo studies in the preceding paper of this issue (Ryan, M. J. (1976), Biochemistry 15). Highly purified Escherichia coli DNA polymerase III and RNA polymerase both were inhibited by this antibiotic. However, the high concentrations necessary for these inhibitions suggest that they are not biologically relevant. No interaction between DNA and coumermycin A1 was observed with the following analytical procedures: ultraviolet difference spectra, DNA absorbance-temperature transitions, equilibrium buoyant density centrifugation, and DNA cross-linking determinations.
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PMID:Coumerimycin A1: A preferential inhibitor of replicative DNA synthesis in Escherichia coli. II. In vivo characterization. 78 23

Amitrole (3-amino-1,2,4-triazole) inhibits bacterial growth both in Escherichia coli and Salmonella typhimurium at a concentration of 0.5% in minimal medium. Repression of growth already occurs at a concentration of 0.1% of amitrole in this medium. In complete medium the bacteria tolerate concentrations of amitrole as high as 1.7-2.4% before growth ceases. Mutagenicity was tested by differential growth comparisons on E. coli strains W 3110 thy pol A1, defective in DNA polymerase I, and its revertant pol A+. Known mutagens (MMS, NTG, mitomycin C) were used as positive controls. Analogous negative results were also obtained in a revertant test when several trp mutant strains of Salmonella were used.
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PMID:Mutagenicity and toxicity of amitrole. III. Microbial tests. 78 46

Aspects of the Salmonella mutagenesis and Escherichia coli DNA polymerase deficient (pol A1) assay procedures for detecting environmental mutagens are discussed. The chief limitation of the pol A1-- assay involves substances that do not diffuse rapidly in agar. This problem can be overcome by performing the test in suspension. A simple procedure for accomplishing this is described. Although the Salmonella assay is more flexible, under routine conditions it does not respond to several classes of substances which give positive responses in the pol A1- system. For optimal testing, it is recommended that the two microbial assays be used in tandem. The DNA-modifying properties of povidone-iodine for eukaryotic and prokaryotic cells are described. Even though this substance does not display mutagenic properties in the standard Salmonella assay, it does so in suspension culture. The basis of the mutagenic and DNA-modifying properties of povidone-iodine appears to involve the iodination of the cystosine residue of DNA.
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PMID:Mutagenicity and DNA-modifying activity: a comparison of two microbial assays. 79 8

Sedimentation analysis of glycerol-density gradients has shown that freshly purified DNA polymerases A and B (pol A and pol B) of Euglena gracilis have molecular weights of 185,000 (8.7S) and 240,000 (10.3S) respectively. They can aggregate in fresh preparations to give forms of higher molecular weight as shown by gel filtration through Sepharose 6B, but on ageing pol B progressively generates species with sedimentation coefficients of 7.4-7.7S, 6.3-6.5S, 4.8S and finally 3.0S. Pol A apparently behaves in a similar fashion though it is unstable. Exposure of pol A and pol B to high ionic strengths can also cause their breakdown to species with lower sedimentation coefficients. The mitochondrial DNA polymerase is distinct, having a molecular weight of 170,000. It is proposed that pol A and pol B are oligomers of the 3.0S subunit and possibly other dissimilar subunits, with pol B having additional factors conferring upon it its extra catalytic functions.
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PMID:DNA polymerases of Euglena gracilis: heterogeneity of molecular weight and subunit structure. 80 92

The subcellular location and growth stage dependence of the DNA polymerases of Euglena gracilis strain Z and of a bleached derivative of the strain have been studied by fractionation of the enzymes from extracts of whole cells and subcellular fractions on DEAE-cellulose. A new method for the rapid isolation of nuclei was employed. Of the major enzymes, pol A has a predominantly nuclear location and pol B a predominantly cytoplasmic location. Pol A is 4-fold and pol B 15-fold more active in exponentially-growing cells than in stationary-phase cells, pol B representing 90% of the combined activities in exponential-phase cells. The activity of the mitochondrial DNA polymerase increases about 3-fold as the cells enter stationary phase while that of the chloroplast DNA polymerase is greater in exponential-phase cells. The chloroplast enzyme persists in cells which have been reversibly bleached. The results are compared to those of similar experiments involving primitive and higher eucaryotes.
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PMID:Subcellular location and growth stage dependence of the DNA polymerases of Euglena gracilis. 81 Jun 22

The major DNA polymerase activity of wild-type U. maydis has been extensively purified. It possesses a molecular weight of about 150,000 daltons and appears to require a DNA primer with a 3'-hydroxyl terminus as well as a template. The polymerase activity has also been purified from the pol 1-1 strain, which is temperature sensitive fro growth and DNA synthesis, and which at the restrictive temperature contains only 10-25% levels of the DNA polymerase activity obtained from wild-type strains. It was similar in all properties studied, except that the activity was thermolabile at 40 degrees C compared to that from the wild-type strain. Physiological studies on the mutant showed that it was only slightly sensitive to UV, ionising radiation and nitrosoguanidine at the permissive temperature, and was proficient in genetic recombination. The results suggest that the pol 1-1 gene product does not play an important role in repair and recombination processes within the cell, and that its primary function lies in replication.
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PMID:DNA polymerase of Ustilago maydis: partial characterization of the enzyme and a pol 1 mutation. 122 4

The action of 5-trifluoromethyl-2'-deoxyuridine (CF3dUrd) on DNA synthesis was investigated in vitro assay systems with purified DNA polymerases. CF3dUrd was incorporated into the DNA of mammalian cells in culture. We studied the incorporation of CF3dUrd 5'-triphosphate (CF3dUTP) into DNA and effect of CF3dUrd residue on DNA synthesis. Therefore, we synthesized oligonucleotides that allow site specific introduction of a CF3dUrd residue into a synthetic DNA oligonucleotide. After CF3dUTP incorporation, the primer was extended for human DNA polymerase alpha (pol. alpha). When CF3dUrd residue was located at an internucleotide site in the template, however, pol. alpha was exhibited a strong arrest band one nucleotide after the CF3dUrd residue site, and Escherichia coli polymerase I (Klenow fragment) also exhibited a weaker arrest band one nucleotide before the CF3dUrd residue. These results suggested that a mechanism of antitumor activity of CF3dUrd is inhibition of DNA replication.
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PMID:Action of 5-trifluoromethyl-2'-deoxyuridine on DNA synthesis. 128 14

Initiation of Adenovirus DNA replication in vitro requires the presence of three viral proteins (pTP, pol, DBP) and two cellular transcription factors, NFI and Oct-1, that stimulate replication more than 100-fold. NFI assists in binding and positioning of the DNA polymerase in the origin whereas Oct-1 changes the structure of origin DNA. Optimal templates contain, in addition to origin sequences, the covalently bound viral terminal protein (TP). This terminal protein stimulates the template activity over 20 fold compared to protein-free templates. To study the way in which TP exerts its function in vitro we devised a novel method to isolate and label a short origin containing fragment in which the TP was bound in a functional form. This fragment replicated very efficiently and could be used for studying the binding of other replication proteins. Employing alpha-chymotrypsin digestion we show that for enhancement of replication in vitro only a small part of TP is required.
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PMID:Adenovirus DNA replication: the function of the covalently bound terminal protein. 129 Dec 41


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