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)

We measured the in vivo incorporation of 2-aminopurine into DNA of T4 bacteriophage allelic for gene 43 (DNA polymerase), mutator (L56), 43+, and antimutator (L141). The magnitude of incorporation (mol/mol of Thy) was 1/1500 in L56, 1/1600 in 43+, and 1/8900 in L141. The incorporation ratio L56:43+:L141 in vivo was equal to that mediated by the purified DNA polymerases of these allelic phages in vitro. A model for 2-aminopurine-induced A-T in equilibrium G-C transitions is discussed. The model is used to predict the magnitudes of replication errors (C mispairing with a template 2-aminopurine) and incorporation errors (2-aminopurine mispairing with a template C) per round of replication and to investigate the asymmetry in 2-aminopurine-induced transitions favoring the A-T leads to G-C pathway over G-C leads to A-T. We suggest that the fidelity of L56 and L141 DNA polymerases exemplifies one-step and two-step editing, respectively.
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PMID:2-Aminopurine-induced mutagenesis in T4 bacteriophage: a model relating mutation frequency to 2-aminopurine incorporation in DNA. 27 Jul 13

The fidelity of DNA synthesis as determined by the misincorporation of the base analogue 2-aminopurine in competition with adenine has been measured as a function of deoxynucleoside triphosphate substrate concentrations using purified mutator (L56), antimutator (L141), and wild type (T4D) T4 DNA polymerases. Although the rates of both incorporation and turnover of aminopurine and adenine decrease as substrate concentrations are decreased, the ratio of turnover/polymerase activity is increased. Thus, the nuclease/polymerase ratio of each of these three DNA polymerases can be controlled. The misincorporation of aminopurine decreases with decreasing substrate concentrations such that all three enzymes approach nearly identical misincorporation frequencies at the lowest substrate concentration. The increased accuracy of DNA synthesis corresponds to conditions producing a high nuclease/polymerase ratio. The misinsertion frequency for aminopurine is independent of substrate concentrations and enzyme phenotype; therefore, the increased accuracy of DNA synthesis with decreasing substrate concentrations is shown to be a result of increased nuclease activity and not increased polymerase or nuclease specificity. The data are analyzed in terms of a kinetic model of DNA polymerase accuracy which proposes that discrimination in nucleotide insertion and removal is based on the free energy difference between matched and mismatched base pairs. A value of 1.1 kcal/mol free energy difference, delta G, between adenine: thymine and aminopurine:thymine base pairs is predicted by model analysis of the cocentration dependence of aminopurine misincorporation and removal frequencies. An independent estimate of this free energy difference based on the 6-fold higher apparent Km of T4 DNA polymerase for aminopurine compared to adenine also gives a value of 1.1 kcal/mol. It is shown that the aminopurine misinsertion frequency for an enzyme having either extremely low 3'-exonuclease activity, Escherichia coli DNA polymerase I, or no measurable exonuclease activity, calf thymus DNA polymerase alpha, is 12 to 15%, which is similar to that for the T4 polymerases and consistent with delta G approximately 1.1 kcal/mol.
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PMID:Error induction and correction by mutant and wild type T4 DNA polymerases. Kinetic error discrimination mechanisms. 42 61

Spontaneous, 2-aminopurine- and 5-bromouracil-induced mutations at six rII nonsense codons were studied in phage T4 strains possessing wild-type and mutant gene 43 alleles. The mutation pathways studied included interconversions and reversions of nonsense codons. The tsCB87 allele, which specifies an antimutator DNA polymerase, reduced base-analogue-induced mutation frequencies along all pathways. However, GC base pairs were less affected than AT base pairs. The frequency of spontaneous UAA leads to UAG conversions was also reduced by tsCB87, but that of spontaneous UAA leads to UAG UGA conversions was often increased. Mutation in the presence of the mutator allele tsL56 was increased along all pathways, with no preference for either AT or GC base pairs. Mutation frequencies in the presence of the two mutant DNA polymerases were highly variable. A strong correlation was found between 2-aminopurine-induced mutation frequencies in ts+ tsCB87 phage along the reversion and UAA changed to UAG (but not UAA changed to UGA) pathways.
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PMID:Site specificity and variability in the mutator and antimutator effects of phage T4 gene 43 mutants. 74 72

Adenine residues in DNA are oxidized under the action of ionizing radiation at the C-8 position to give 7,8-dihydro-8-oxoadenine. The formation of this lesion can be considered a cause of mutations and carcinogenesis. Oligodeoxyribonucleotides 39 and 47 bases long containing a single 7,8-dihydro-8-oxoadenine (8-hydroxyadenine) residue were synthesized by using nucleoside phosphoramidites. They were used as templates to study the copies obtained in vitro by the Klenow fragment and the thermostable Taq DNA polymerase. 7,8-Dihydro-8-oxoadenine does not block the replication and thymine is incorporated opposite the damage. The modifications of the DNA duplex conformation provoked by 7,8-dihydro-8-oxoadenine are minor. 1H-NMR spectroscopy shows that the duplex is in a B form, the sugar in a normal position in the helix and the modified base in the anti position. NMR confirms that 7,8-dihydro-8-oxoadenine exists predominantly in the keto form.
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PMID:Structure and in vitro replication of DNA templates containing 7,8-dihydro-8-oxoadenine. 185 59

The current progress in antiviral therapy is related to our better understanding of the viral multiplication, with potential targets for specific antiviral action at each step of the multiplication cycle inside the infected cell. Amantadine and Rimantadine are anti-influenza A drugs interfering with the penetration and the release of the virus. Most of the other antiviral drugs which are clinically available have the same target in common, namely the viral DNA polymerase. This holds true for modified nucleosides such as Acycloguanosine (Acyclovir), DHPG, Adenine-Arabinoside, Azidothymidine as well as pyrophosphate derivatives such as phosphonoformic acid. Unfortunately the antiviral chemotherapy must confront 3 obstacles: 1) a possible interference with the normal cellular metabolism, leading to residual cytotoxic side effects; 2) the genetic variability of the viruses, producing drug-resistant mutants and 3) the inability of any antiviral chemotherapeutic agent known to date to eradicate latent viral infection. A new approach of the control of latent infection is suggested with anti sense oligonucleotides of hybridons.
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PMID:Perspectives in antiviral chemotherapy. 221 May 92

By promoter fusion to the galK gene and comparative S1 analysis we investigated the in vivo regulation of transcription of the dnaQ gene which encodes the epsilon-subunit of the DNA polymerase III holoenzyme carrying the 3'----5' exonucleolytic proofreading function. Induction of a mutagenic stress situation by treatment with the base analogue 2-aminopurine (2-AP) leads to an increase in dnaQ transcription. S1 mapping analysis of the two dnaQ transcripts revealed a differential promoter activation for this 2-AP induced increase in dnaQ transcription. In addition, a similar galK promoter fusion with the dnaN gene coding for the beta-subunit of the DNA polymerase III holoenzyme revealed that dnaN transcription is also 2-AP inducible as judged by galactokinase activity. This is the first evidence for the inducibility of dnaQ gene expression (and possibly of other genes of the DNA polymerase II holoenzyme) and is discussed in relation to DNA repair mechanisms.
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PMID:Expression of the Escherichia coli dnaQ (mutD) gene is inducible. 283 Apr 59

We propose a model to describe the frequencies of site-specific base substitution errors by DNA polymerase. In the model, nucleotide misinsertion frequencies are determined by 5'-nearest-neighbor base stacking and 3'-exonucleolytic proofreading efficiencies are governed by the relative proportions of G . C base pairs in the region surrounding the misinserted nucleotide. The model is used to analyze the frequency of replacing dAMP by 2-aminopurine deoxyribonucleotide with purified bacteriophage T4 L141 antimutator DNA polymerase at 57 sites on phi X174 DNA (Pless, R. C., and Bessman, M.J. (1983) Biochemistry 22, 4905-4915). A linear least-squares fit yields a correlation coefficient of 0.83 and a standard deviation of 2.8% between predicted and observed results. Four to five base pairs on each side of the 2-aminopurine incorporation site, approximately one double-helical turn, are found to exert a maximal influence on proofreading. Thermal melting data on native and synthetic DNA are used to deduce base-stacking energies for nearest-neighbor doublets including those involving 2-aminopurine. The inclusion of base-stacking energies in the model calculations leads to predictions similar to those based on the use of empirical parameters for individual base pairs.
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PMID:Influence of neighboring bases on DNA polymerase insertion and proofreading fidelity. 315 58

DNA heptamers containing the mutagenic base analogue 2-aminopurine (AP) have been chemically synthesized and physically characterized. We report on the relative stabilities of base pairs between AP and each of the common DNA bases, as determined from heptamer duplex melts at 275 and 330 nm. Base pairs are ranked in order of decreasing stability: AP.T greater than AP.A greater than AP.C greater than AP.G. It is of interest that AP.A is more stable than AP.C even though DNA polymerase strongly favors the formation of AP.C over AP.A base pairs. Comparisons of melting profiles at 330 nm and 275 nm indicate that AP.T, AP.A, and AP.C base pairs are annealed in heptamer duplexes and melt 2-3 degrees prior to surrounding base pairs, whereas AP.G appears not to be annealed.
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PMID:Synthesis and properties of defined DNA oligomers containing base mispairs involving 2-aminopurine. 373 16

Ribonucleoside and deoxyribonucleoside triphosphate pools have been measured in Escherichia coli infected with bacteriophage T4 DNA polymerase mutator, wild type, and antimutator alleles during mutagenesis by the base analogue 2-aminopurine. ATP and GTP pools expand significantly during mutagenesis, while CTP and UTP pools contract slightly. The DNA polymerase (gene 43) alleles and an rII lesion perturb normal dNTP pools more than does the presence of 2-aminopurine. We find no evidence that 2-aminopurine induces mutations indirectly by causing an imbalance in normal dNTP pools. Rather, it seems likely that, by forming base mispairs with thymine and with cytosine, 2-aminopurine is involved directly in causing bidirectional A.T in equilibrium G.C transitions. The ratios for 2-aminopurine deoxyribonucleoside triphosphate/dATP pools are 5-8% for tsL56 mutator and 1-5% for tsL141 antimutator and 43+ alleles. We conclude that the significant differences observed in the frequencies of induced transition mutations in the three alleles can be attributed primarily to the properties of the DNA polymerases with their associated 3'-exonuclease activities in controlling the frequency of 2-aminopurine.cystosine base mispairs.
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PMID:Ribonucleoside and deoxyribonucleoside triphosphate pools during 2-aminopurine mutagenesis in T4 mutator-, wild type-, and antimutator-infected Escherichia coli. 388 83

Adenine residues of 70S avian myeloblastosis virus (AMV) RNA are modified when reacted with chloroacetaldehyde. This modification introduces characteristic fluorescent epsilon-adenosine (epsilonA) probes which were used to monitor the reaction. Under suitable conditions, modified 70S(epsilonA) RNA was maintained intact and was inactive as a template for the AMV DNA polymerase. Furthermore, it inhibited the reaction catalyzed by AMV polymerase when 70S RNA was used as template-primer and had no effect on the two tested bacterial polymerases. Protection against the 70S (epsilonA) RNA inhibition was observed when 70S RNA was primed with oligo(dT) indicating preference of the polymerase for the oligo(dT) primed regions.
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PMID:Effect of chemically modified 70S RNA from avian myeloblastosis virus (AMV) upon the activity of AMV DNA polymerase. 437 90


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