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)

Escherichia coli strains containing mutations in various deoxyribonucleic acid synthesis cistrons have been tested for their ability to support bacteriophage N4 growth and, specifically, N4 DNA synthesis. N4 DNA synthesis is independent of the activity of the products of the E. coli dnaA, dnaB, dnaC, dnaE, dnaG, and rep genes. In contrast, N4 DNA replication requires the products of the dnaF, (ribonucleotide reductase) and lig (DNA ligase) genes of E. coli. N4 DNA replication, specifically processing of short DNA fragments requires the 5'-3' exonuclease activity of the polA gene product. However, its DNA polymerizing activity is not required. In addition, the sensitivity of N4 DNA synthesis to inhibitors or temperature-sensitive mutants of E. coli DNA gyrase suggests that this activity is required for N4 DNA synthesis. To date, we have found five N4 gene products required for N4 DNA replication: dbp (a single-stranded DNA binding protein), dnp (a DNA polymerase), dns (unknown function), vRNAp (the N4 virion-associated, DNA-dependent RNA polymerase) and exo (a 5'-3' exonuclease).
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PMID:Host and phage-coded functions required for coliphage N4 DNA replication. 300 44

Recently, 2-halogenated deoxyadenosine analogs (F, Cl, and Br) have been shown to have antitumor activity. These analogs are phosphorylated by cells and are believed to exert their cytotoxic action at the nucleoside triphosphate level. In this work the interaction of these nucleoside triphosphate analogs with potential targets, such as DNA polymerase alpha, beta, and gamma, DNA primase, and ribonucleotide reductase was examined in detail. All of these compounds competitively inhibited the incorporation of dAMP into DNA by DNA polymerase alpha, beta, or gamma. F-dATP was able to completely substitute for dATP using DNA polymerase alpha and gamma, but not with DNA polymerase beta. Cl-dATP and Br-dATP substituted poorly for dATP using DNA polymerase alpha and beta. Extension of a 32P-labeled primer by DNA polymerase alpha, beta, or gamma on a single-stranded M13 template showed that these compounds were incorporated into the 3' end of the growing DNA chain and that elongation beyond the incorporated analogs was significantly retarded for Cl-dATP and Br-dATP using either DNA polymerase alpha or beta. DNA primase using poly(dC) as template was inhibited by these compounds at a concentration 4 to 5 times greater than that required for 2-F-araATP. The 2-halogenated dATP analogs were potent inhibitors of ADP reduction by ribonucleotide reductase. In conclusion, the cytotoxic action of 2-Cl-deoxyadenosine and 2-Br-deoxyadenosine may partially be mediated through the mechanism of "self-potentiation," by depression of the deoxynucleoside triphosphate pools due to inhibition of ribonucleotide reductase, which would facilitate their incorporation into DNA and result in the inhibition of DNA synthesis.
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PMID:Interaction of 2-halogenated dATP analogs (F, Cl, and Br) with human DNA polymerases, DNA primase, and ribonucleotide reductase. 305 Apr 47

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

Thioredoxin is a small (Mr 12,000) ubiquitous redox protein with the conserved active site structure: -Trp-Cys-Gly-Pro-Cys-. The oxidized form (Trx-S2) contains a disulfide bridge which is reduced by NADPH and thioredoxin reductase; the reduced form [Trx(SH)2] is a powerful protein disulfide oxidoreductase. Thioredoxins have been characterized in a wide variety of prokaryotic cells, and generally show about 50% amino acid homology to Escherichia coli thioredoxin with a known three-dimensional structure. In vitro Trx-(SH)2 serves as a hydrogen donor for ribonucleotide reductase, an essential enzyme in DNA synthesis, and for enzymes reducing sulfate or methionine sulfoxide. E. coli Trx-(SH)2 is essential for phage T7 DNA replication as a subunit of T7 DNA polymerase and also for assembly of the filamentous phages f1 and M13 perhaps through its localization at the cellular plasma membrane. Some photosynthetic organisms reduce Trx-S2 by light and ferredoxin; Trx-(SH)2 is used as a disulfide reductase to regulate the activity of enzymes by thiol redox control. Thioredoxin-negative mutants (trxA) of E. coli are viable making the precise cellular physiological functions of thioredoxin unknown. Another small E. coli protein, glutaredoxin, enables GSH to be hydrogen donor for ribonucleotide reductase or PAPS reductase. Further experiments with molecular genetic techniques are required to define the relative roles of the thioredoxin and glutaredoxin systems in intracellular redox reactions.
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PMID:Thioredoxin and related proteins in procaryotes. 315 90

We have studied the antiproliferative effects of gallium nitrate in cultured CCRF-CEM lymphoblasts. The 50% inhibitory dose for these cells was 120 microM, and after 24 h at a cytostatic concentration (480 microM) S-phase arrest was observed by DNA flow cytometry. Deoxyribonucleoside triphosphate pools were all reduced (dATP, dGTP, and dCTP by 50%, dTTP by 25%), suggesting inhibition of ribonucleotide reductase. Administration of tracer amounts (0.5 microM) of either [3H]uridine or [3H]deoxyuridine confirmed that DNA synthesis had been inhibited to 20% of control rates by gallium. Further, the flow of the ribonucleoside into the dTTP pool and DNA was selectively reduced compared to that of the deoxyribonucleoside. Gallium decreased the specific activity of dTTP labeled from uridine by 50%, whereas the specific activity of dTTP labeled from deoxyuridine was increased 2.5-fold. Thus counts in DNA derived from [3H]uridine were decreased by more than 80%, while counts in DNA derived from [3H]deoxyuridine were virtually unaltered. Uridine incorporation into RNA was not affected. Gallium did not significantly alter the capacity of permeabilized naive cells to incorporate [3H]dTTP into DNA, while 24-h gallium pretreatment (which increased the percentage of S-phase cells) produced a modest increase in [3H]dTTP incorporation, indicating that any effect of gallium on DNA polymerase alpha is minor. Gallium treatment did not induce or inhibit the repair of DNA single strand breaks. These data demonstrate that gallium inhibits replicative DNA synthesis, with the major specific enzyme target probably being ribonucleotide reductase.
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PMID:Effect of gallium on DNA synthesis by human T-cell lymphoblasts. 325 58

Coliphage N4 replication is independent of most host DNA replication functions except for the 5'----3' exonuclease activity of polA, DNA ligase, DNA gyrase, and ribonucleotide reductase (Guinta, D., Stambouly, J., Falco, S. C., Rist, J. K., and Rothman-Denes, L. B. (1986) Virology 150, 33-44). It is therefore expected that N4 codes for most of the functions required for replication of its genome. In this paper we report the purification of the N4-coded DNA polymerase from N4-infected cell extracts by following its activity on a gapped template and in an in vitro complementation system for N4 DNA replication (Rist, J. K., Pearle, M., Sugino, A., and Rothman-Denes, L. B. (1986) J. Biol. Chem. 261, 10506-10510). The enzyme is composed of one polypeptide, Mr 87,000. It is most active on templates containing short gaps synthesizing DNA with high fidelity in a quasi-processive manner. A strong 3'----5' exonuclease activity is associated with the DNA polymerase polypeptide. No 5'----3' exonuclease or strand-displacing activities were detected.
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PMID:Purification and characterization of bacteriophage N4-induced DNA polymerase. 340 28

Using partially purified enzyme from L1210 cells, dihydroxybenzene derivatives related structurally to dopamine were shown to reversibly inactivate ribonucleotide reductase. A structure-activity analysis revealed that derivatives with side-chains, which contain a negatively-charged group, had significantly reduced inhibitory activity. The ability of these compounds to inhibit ribonucleotide reductase was dependent on the hydroxyl groups being in the ortho position and did not correlate with free radical inhibitory activity. A kinetic analysis by the method of Lineweaver-Burk indicated that the inhibition of ribonucleotide reductase by the derivative 3,4-dihydroxybenzylamine was competitive with the reducing substrate dithioerythritol. This analog, in combination with hydroxyurea, gave synergistic inhibition or ribonucleotide reductase, suggesting different sites of action. Using Tween 80-treated L1210 cells, it was found that these drugs had an immediate inhibitory effect on ribonucleotide reductase activity in intact, reversibly permeabilized cells. Furthermore, although these drugs had no immediate effect on DNA polymerase, in permeabilized L1210 cells (when the cells were preincubated with the dihydroxybenzene derivatives for 1 hr prior to permeabilization), there was significant inhibition of DNA polymerase activity. The two key enzymes for DNA synthesis appear to be sequentially inhibited by these analogs, with the reduced form (quinol) inhibiting ribonucleotide reductase and the oxidized form (quinone) inhibiting DNA polymerase.
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PMID:Inhibition of ribonucleotide reductase by antitumor agents related to levodopa and dopamine. 388 92

Recent studies in our laboratory and others have demonstrated that DNA polymerase inhibitors such as the ara nucleosides, aphicolin and dideoxythymidine are potent inhibitors of the DNA excision repair process in confluent human fibroblasts as evidenced by the agent-dependent accumulation of single-strand interruptions in the DNA of UV-irradiated, but not in unirradiated, cellular DNA. In rapidly cycling cells, on the other hand, these agents are weak inhibitors at best but when used in combination with the ribonucleotide reductase inhibitor, hydroxyurea, a significant enhancement of inhibitory capacity is seen. In an attempt to better understand the mechanism of repair inhibition by DNA polymerase inhibitors, and the nature of this hydroxyurea enhancement, experiments were initiated in which the effects of a series of ribonucleotide reductase inhibitors on dNTP pools and on the DNA repair process were determined in both quiescent cultures and log-phase cultures of human fibroblasts. It was determined that hydroxyurea, deoxyadenosine, pyridine-2-carboxaldehyde thiosemicarbazone (TSC), pyrozoloimidazole (IMPY), 3,5-diamino-1,2,4-triazole (guanazole), 3,4,5-trihydroxy benzohydroxamic acid (THBA) and 3,4-dihydroxy benzohydroxamic acid (DHBA) are all effective inhibitors of the DNA repair process in confluent cells but not in log-phase cells. Moreover, the effects of these inhibitors can be reversed by the addition of certain combinations of deoxynucleosides. These reversal studies and the direct analysis of dNTP pool modulation by these compounds in log phase and confluent cultures support the notion that specific pool depletions rather than general imbalance of pools gives rise to the inhibition of the DNA excision repair process.
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PMID:Effects of nucleotide pool imbalances on the excision repair of ultraviolet-induced damage in the DNA of human diploid fibroblasts. 388 72

Analysis of ribonucleotide reductase and DNA polymerase activities in extracts of Pseudomonas stutzeri by centrifugation in discontinuous sucrose gradients indicated that these two enzymes are associated with two different high molecular weight cellular components. In addition, 95% of the ribonucleotide reductase activity was pelleted by centrifugation of extracts for 4 hr at 200,000 X G. The reductase activity remained particulate (sedimentable) following sonication whereas some 90% of the DNA polymerase activity was rendered soluble (non-sedimentable) by this technique. This data indicate that the P. stutzeri ribonucleotide reductase is not a cytosolic enzyme, but is associated with a macromolecular component in the cell.
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PMID:Sedimentation analysis of ribonucleotide reductase activity in extracts of Pseudomonas stutzeri. 388 16

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


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