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)

Treatment of growing cultures of Mycobacterium smegmatis with alkylating agents (methyl methanesulphonate, ethyl methanesulphonate, nitrogen mustard, or mitomycin C) or with ultraviolet light resulted in enhanced specific activities of a DNA polymerase and of an ATP-dependent deoxyribonuclease. Similar results had previously been obtained with hydroxyurea and with iron limitation. The three of these treatments which were tested (methyl methanesulphonate, mitomycin C and hydroxyurea) produced strand breaks or alkali-labile regions in the DNA of this organism. The increased enzyme activities could be prevented by simultaneous treatment with inhibitors of protein synthesis. In contrast, treatment of the cultures with intercalating agents (ethidium bromide, acridine orange, or proflavine), 5-fluorouracil, caffeine, or nalidixic acid, inhibited DNA synthesis without increasing the enzyme activities. These treatments did not produce strand breaks in the DNA of this organism. The results support the hypothesis that, in M. smegmatis, damage to DNA induces increased synthesis of enzymes associated with DNA repair.
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PMID:Increased DNA polymerase and ATP-dependent deoxyribonuclease activities following DNA damages in mycobacterium smegmatis. 84 85

A single dose of erythropoietin stimulates DNA synthesis in the spleen of the polycythemic mouse with the maximum effect occurring 48 h after the hormone is administered. The increase in DNA synthesis is accompanied by morphologic evidence of increased erythropoiesis and by increases in the activities per cell of both thymidine kinase and cytoplasmic high molecular weight DNA polymerase-alpha. The activity of low molecular weight DNA polymerase-beta does not change significantly. Spleen cells from mice which had received either erythropoietin or saline 48 h previously were separated into 7 density classes on discontinuous bovine serum albumin gradients. Following the administration of erythropoietin, thymidine incorporation and thymidine kinase activity showed the greatest relative increases per nucleated cell in layers 3, 4 and 5 of the gradient. DNA polymerase-alpha showed the greatest increase in cells of the denser layers 5, 6 and 7. Each layer contained normoblasts and lymphocytes. The less well differentiated erythroid elements constituted a larger proportion of cells in layers of lower density. Increases in the rates of thymidine incorporation were better correlated with increases in thymidine kinase activity than with increases in DNA polymerase activities. Measurement of iron incorporation into heme confirm the morphological impression that the cell type responsible for increased thymidine incorporation and increased DNA polymerase-alpha activity is the young normblast.
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PMID:DNA polymerase, thymidine kinase and DNA synthesis in erythropoietic mouse spleen cells separated on bovine serum albumin gradients. 125 82

Ribonucleotide reductase which catalyzes the rate-limiting step in the de novo synthesis of dNTPs is composed of two non-identical protein subunits which are not under coordinate control in terms of synthesis and degradation. The mRNAs for the effector-binding (EB) and non-heme iron (NHI) subunits are likewise not under coordinate control during cell cycle traverse. Inhibitors directed at the specific subunits of ribonucleotide reductase block DNA synthesis. These current studies show that drugs such as IMPY or hydroxyurea which specifically inhibit the NHI subunit cause a marked increase in the steady-state level of the mRNA for the NHI subunit while resulting in a decrease in the level of mRNA for the EB subunit. In cells treated with deoxyadenosine, the patterns of the mRNAs for the NHI and EB subunits were different from those seen in the IMPY- or hydroxyurea-treated cells. Control experiments utilizing inhibitors (aphidicolin or araC) directed at DNA polymerase showed that the pattern of changes in the mRNA levels for the NHI and EB subunits were specific for the reductase inhibitors. These changes in the mRNAs for the NHI and EB subunits may be due to drug-induced alterations in transcription rates and/or degradation rates for the specific mRNAs.
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PMID:Factors affecting the mRNA levels for the non-heme iron and effector-binding subunits of ribonucleotide reductase. 149 19

1. DNA damage by peplomycin, an antitumor antibiotic, and its repair by cellular enzymes were studied using pUC18 plasmid DNA. The DNA damage and repair were measured by monitoring the conformational changes of pUC18 DNA. 2. Peplomycin-induced DNA damage was enhanced by addition of ferrous ion and inhibited by deferoxamine, a specific iron chelator, suggesting iron-requirement for the DNA damage. 3. DNA damage by peplomycin was inhibited by superoxide dismutase in both native and heat-inactivated forms, possibly due to non-enzymatic interaction. 4. Peplomycin-induced, single-strand breaks in pUC18 DNA was repaired by incubating with a priming factor (an exonuclease purified from mouse ascites sarcoma cells), DNA polymerase beta, four deoxynucleoside triphosphates, T4 DNA ligase and ATP. The average repair patch size was estimated to be approximately four nucleotide length.
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PMID:DNA damage by peplomycin and its repair in an in vitro system. 171 61

The DNA polymerase chain reaction was developed for in vitro amplification of specific DNA sequences, and it has been used for a wide variety of purposes in several fields. We have developed an application of the polymerase chain reaction that is useful for the isolation of partial cDNA or genomic clones of conserved genes. We used this technique to clone the gene encoding the iron protein subunit (27 kDa) of succinate dehydrogenase (EC 1.3.5.1) from several species, including human, rat, Drosophila melanogaster, Arabidopsis thaliana, Schizosaccharomyces pombe, and Saccharomyces cerevisiae. Mixed oligonucleotide primers corresponding to two conserved regions of the protein were used in conjunction with genomic and cDNA templates in the reaction. The primers contained all possible nucleotide combinations that could encode the corresponding peptide sequences. These oligonucleotide mixtures contained 262,144 (2(18] and 8192 (2(13] unique sequences, respectively. Use of the polymerase chain reaction for homology probing allows one to utilize more complex mixtures of oligonucleotides as probes than is possible with filter hybridization screening techniques. In addition, the polymerase chain reaction offers the advantage of synthesizing the DNA product directly, in some cases obviating the need to construct cDNA or genomic libraries. This application of the polymerase chain reaction should be useful not only for the identification of conserved genes in a variety of species but also for the isolation of previously unknown members of gene families.
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PMID:Use of the DNA polymerase chain reaction for homology probing: isolation of partial cDNA or genomic clones encoding the iron-sulfur protein of succinate dehydrogenase from several species. 249 55

Four 25-nt oligonucleotides consisting of sequences of dA and dT (D1-4) have been synthesized. As shown in a companion paper (Rippe et al., 1989), the two combinations D1.D3 and D2.D4 form normal antiparallel duplexes, whereas the pairs D1.D2 and D3.D4 constitute duplexes with the same sequences, but with the two strands parallel to each other. The activities of the following DNA processing enzymes and chemical reagents on the parallel stranded (ps) and antiparallel stranded (aps) duplexes were tested. (i) The restriction endonucleases DraI, SspI, and MseI do not cut the ps duplexes. (ii) DNase I and exonuclease III exhibit a much lower activity with the ps duplexes. (iii) The nuclease activities of S 1 nuclease, micrococcal nuclease (S 7), phage lambda 5'-exonuclease, and the 3'-5' nuclease activity of Escherichia coli DNA polymerase I and its large fragment are higher with the ps than with the aps substrates. (iv) Bal 31 nuclease and the chemical nuclease 1,10-phenanthroline-copper ion [(OP)2Cu+] degrade ps-DNA and aps-DNA at approximately the same rate but show preferred cutting sites only with the aps molecules. (v) The iron(II)-EDTA complex has equivalent nuclease activities with the ps and the aps molecules. (vi) The ps duplex is not a substrate for blunt-end ligation with phage T4 DNA ligase.
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PMID:Substrate properties of 25-nt parallel-stranded linear DNA duplexes. 255 23

Bacteriophage T7 DNA polymerase, the product of gene 5 of the phage, has both polymerase and single-and double-stranded DNA 3'-to 5'-exonuclease activities. The exonuclease activities can be inactivated selectively by an oxidation reaction that requires molecular oxygen, a reducing agent, and iron at a concentration less than or equimolar to that of the gene 5 protein. Both exonuclease activities can be diminished by several thousandfold, with only a small decline in the polymerase activity. Escherichia coli thioredoxin, an accessory protein that binds tightly to the gene 5 protein and increases the processivity of the polymerization reaction, has no effect on the rate of oxidation. We propose that iron binds specifically to the exonuclease domain and, in the presence of molecular oxygen and a reducing agent, generates reactive oxygen species that selectively modify amino acid residues essential for the exonuclease activities.
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PMID:Selective oxidation of the exonuclease domain of bacteriophage T7 DNA polymerase. 282 55

Ribonucleotide reductase, because of the critical role that it plays in DNA replication and the specific properties of the protein subunits, provides a unique metabolic target for chemotherapeutic approaches to cancer treatment. Combinations of ribonucleotide reductase inhibitors resulted in synergistic inhibition of cell growth with concurrent cytotoxicity. The drugs in this combination were targeted at the individual subunits (non-heme iron and effector-binding) of ribonucleotide reductase and at the differential sensitivities of the substrate reductions to these agents. The reduction of the intracellular pools of all four dNTPs through the direct inhibition of ribonucleotide reductase has the effect of reducing DNA polymerase activity in a sigmoidal manner rather than in a hyperbolic fashion due to the requirement of DNA polymerase for all four substrates. As a result relatively small decreases in the intracellular concentrations of the dNTPs cause remarkably large decreases in DNA synthesis and hence cell replication. It appears that there may be a relationship between the capability of the cell to synthesize DNA at a minimal absolute rate and cell viability. That is, if DNA synthesis is decreased to or below a specific level, then the processes leading to cell death takes precedence over the tendency of the cell to complete DNA replication leading to cell division.
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PMID:Ribonucleotide reductase as a chemotherapeutic target. 307 32

Ribonucleotide reductase is a key enzyme in DNA replication and, as such, has been a target for antitumor agents. This enzyme is composed of two nonidentical protein subunits which can be specifically and independently inhibited. Combinations of drugs directed at the effector-binding and non-heme iron subunits of ribonucleotide reductase resulted in the synergistic inhibition of L1210 cell growth and synergistic L1210 cell kill. These combinations included dAdo/EHNA/IMPY/Desferal; dAdo/EHNA/hydroxyurea/Desferal (the EHNA was required to protect dAdo from deamination while Desferal modulated the effects of IMPY or hydroxyurea); 2-F-araA/IMPY/Desferal and 2-F-2'-dAdo/IMPY/Desferal (EHNA was not required to protect 2-F-araA or 2-F-2'-dAdo from deamination); and dGuo/8-AGuo/IMPY/Desferal (8-AGuo was required to protect dGuo from phosphorolysis). Although thymidine alone inhibited L1210 cell growth, it was not possible to potentiate the effects of thymidine with the pyrimidine nucleoside phosphorylase inhibitors, acyclothymidine, 5-chlorouracil and 2,6-dihydroxypyridine. Combinations of drugs directed at the ribonucleotide reductase and DNA polymerase sites were studied for their effects on L1210 cell growth. With these combinations, no synergistic inhibition of L1210 cell growth was observed. The combinations of aphidicolin and IMPY/Desferal and aphidicolin and dAdo/EHNA inhibited L1210 cell growth in an additive manner; the combinations of IMPY/Desferal and BuAU or IMPY/Desferal and BuPdG resulted in antagonistic inhibition of L1210 cell growth. From these results it is clear that combination chemotherapy directed at independent sites of the same key target enzyme can result in strong synergistic inhibition of cell growth and cytotoxicity offering a clear therapeutic advantage. In contrast, the combinations directed at sequential key enzymes (e.g. ribonucleotide reductase and DNA polymerase) did not result in synergistic inhibition of cell growth. The utility of combinations of drugs directed at specific but independent sites of the target enzyme (e.g. ribonucleotide reductase) has been demonstrated in tumor cell systems in culture and now must be demonstrated in vivo.
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PMID:The utility of combinations of drugs directed at specific sites of the same target enzyme--ribonucleotide reductase as the model. 390 3

Ribonucleotide reductase catalyzes the rate-limiting step in DNA synthesis. It represents a key metabolic site at which specific inhibitors have been directed as potential antitumor agents. Several different classes of ribonucleotide reductase inhibitors have been generated and studied. Because of the nature of the DNA polymerase reaction in which all four dNTPs are required, the initial velocity vs dNTP concentration curve gives sigmoidal rather than hyperbolic kinetics. As a result, a 50 per cent decrease in ribonucleotide reductase activity causes a decrease in DNA polymerase activity of 75 per cent or greater depending on the ratio of [dNTP] to its Km. This has been demonstrated with theoretical calculations, actual DNA polymerase determinations and precursor studies in intact tumor cells. The structural requirements for a compound to serve as a specific inhibitor of ribonucleotide reductase, either as the non-heme iron or effector-binding subunit, are stringent. Each protein subunit comprising the active enzyme can be specifically and independently inhibited. When combinations of agents, each directed at one of the subunits of ribonucleotide reductase, are used, strong synergistic inhibition of L1210 cell growth and synergistic cytotoxicity result.
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PMID:Drug action on ribonucleotide reductase. 391 89


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