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:2.7.7.49 (reverse transcriptase)
31,746 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Phosphonoacetate is a highly specific inhibitor of herpes simplex virus-induced DNA polymerase. Sensitivity of herpesvirus type 1 or type 2 induced DNA polymerase to the drug was similar. However, DNA polymerases from other sources such as the host cells (Wi-38), Micrococcus luteus, and hepatitis B virus were highly resistant. In addition, Escherichia coli RNA polymerase and reverse transcriptase of Rous sarcoma virus were also insensitive to the drug. Enzyme kinetic studies showed that inhibition was noncompetitive with respect to deoxyribonucleotide triphosphates. The Ki value was about 0.45 muM. The apparent Km values for dTTP, dATP, dCTP, and dGTP were 0.71, 0.75, 0.42, and 0.39 muM, respectively. The base composition of template has no profound effect on the extent of inhibition. The drug caused uncompetititve inhibition with respect to template which indicated that phosphonoacetate did not bind directly to template DNA. Results are presented which suggest that phosphonoacetate did not affect the formation of the enzyme-DNA complex but probably inhibited the elongation step of DNA polymerase reaction.
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PMID:Mode of inhibition of herpes simplex virus DNA polymerase by phosphonoacetate. 5 71

The synthesis of a complementary DNA copy (cDNA) of hen ovalbumin mRNA using AMV RNA-directed DNA polymerase was studied under different conditions of salt, deoxyribonucleotide concentrations, temperature, and time. It was observed that in the absence of monovalent cation at 46 degrees C a complete transcript of ovalbumin mRNA could be effected by the enzyme. The minimum deoxyribonucleotide requirement for complete synthesis was 35 muM for dATP, dGTP, and dCTP and 200 muM dTTP. By a number of different experimental criteria which included sedimentation on alkaline sucrose gradients and electrophoresis in polyacrylamide gels containing 98% formamide, direct electron microscope visualization, and protection of ovalbumin [25I]mRNA from nuclease digestion it could be demonstrated that a considerable fraction of a complete mRNA transcript was indeed synthesized. The cDNA/ovalbumin mRNA hybrid had a Tm on hydroxylapatite of 92 degrees C, indicating the synthesis of a RNA transcript with a high fidelity. When such a complete ovalbumin [3H]cDNA was synthesized with a specific activity of 10(8) cpm/mug and hyfridized to an excess of chick DNA, the kinetics of hybridization indicated that the cDNA was comprised of a nonrepetitive sequence.
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PMID:The synthesis and properties of the complete complementary DNA transcript of ovalbumin mRNA. 5 72

We have measured reverse transcriptase enzyme activity per virus particle for samples of avian myeloblastosis virus (BAI strain) and murine leukemia virus (RAUSCHER) USing the synthetic template poly(rC)-oligo(dG). Absolute virus concentrations were determined directly by laser beat frequency spectroscopy. Enzyme activity per virion was determined from the slope of the activity plotted as a function of virus concentration. With this reverse transcriptase assay, the minimum activity (expressed as picomoles of dGTP incorporated/virion per hour) is estimated at (28.1 +/- 4.2) X 10(-7) for avian myeloblastosis virus and (1.1 +/- 0.2) X 10(-7) for murine leukemia virus. The sensitivity of this assay, which is determined by the level of incorporated radioactivity measurable above background, is 2.5 X 10(-4) virions for avian myeloblastosis virus (with dGTP specific activity of 8.9 Ci/mmol) and 88 X 10(-4) virions for murine leukemia virus (with dGTP specific activity of 6.52 CI/mmol). These results show that although reverse transcriptase assays can obviously be used to measure relative virus concentrations of equally purified samples of the same virus, they can be very misleading when used to compare the concentrations of different virus species.
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PMID:Reverse transcriptase activity per virion for avian myeloblastosis virus and Rauscher murine leukemia virus. 5 65

The alpha, beta2, and alphabeta forms of the RNA-dependent DNA polymerase of avian sarcoma virus B77 grown in duck embryo fibroblasts have been compared with respect to several kinetic properties. The following results were obtained. 1. The Km values for dTTP and dGTP for enzyme forms alpha, beta2, and alphabeta were 77, 39, and 74, and 6.8, 3.1, and 6.1 micronM, respectively. 2. The affinity of 70 S Rous sarcoma virus RNA for enzyme form alphabeta was about twice that for the other two forms. 3. The relative specific activities of the three enzyme forms on synthetic primer-templates such as poly(rA)-poly(dT) were almost the same. The viral 70 S RNA-dependent specific activities were 2 to 3 orders of magnitude lower and in the ratio of 1:3:5 for enzyme forms alpha:beta2:alphabeta. Addition of exogenous oligo(dT) stimulated the 70 S viral RNA-dependent activity of enzyme forms alphabeta and beta2 by a factor of 3, and that of enzyme form alpha by a factor of 30, so that it then became the most active transcriptase of viral 70 S RNA. 4. The largest transcripts formed by the three enzyme forms with 70 S viral RNA as primer-template were about 4,500 nucleotides long. About one-third of the total amount of polynucleotides polymerized by the alphabeta enzyme was in the form of such transcripts. This proportion was far higher than for the other two enzyme forms. 5. All three enzyme forms were capable of transcribing single-stranded into double-stranded DNA. 6. The 3-propylcyclohexyl piperidyl derivative of rifamycin SV, at a concentration of 100 microng/ml, inhibited enzyme forms beta2 and alphabeta by over 99.5 and 96%, respectively, but enzyme form alpha by only about 60%. 7. The beta2 and alphabeta forms of the enzyme were processive DNA polymerases, the alpha form a nonprocessive polymerase. 8. In general, these results indicate that in most respects the properties of the dimeric enzyme forms resemble each other much more closely than those of the alpha form. In some very important respects, such as affinity for viral RNA and the size of transcripts formed from it, the alphabeta enzyme form performs significantly better than either of the other two enzyme forms.
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PMID:RNA-dependent DNA polymerase of avian sarcoma virus B77. II. Comparison of the catalytic properties of the alpha, beta2, and alphabeta enzyme forms. 6 35

Poly(A)-containing encephalomyocarditis virus RNA functions as an excellent template for cDNA synthesis in vitro with an RNA-dependent DNA polymerase in the presence of an oligothymidylate primer. Under appropriate conditions, discrete transcripts of increasing chain length were obtained, suitable for sequence analysis. A limited cDNA fragment of 36 nucleotides, primer (dT)10 included, was synthesized when dGTP was omitted from the reaction mixture and its primary structure was elucidated using direct DNA-sequencing methods. The complement corresponds to the 3' end of encephalomyocarditis RNA. The hexanucleotide (5'-3')(A-A-U-A-A-A) found in this sequence is also present in all 3' non-coding regions of poly(A)-containing eukaryotic mRNAs studied until now, in nearly identical positions relative to the poly(A) tail. The possible biological significance of this structural homology is discussed.
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PMID:The 3'-Terminal nucleotide sequence of encephalomyocarditis virus RNA. 7 34

In an effort to better understand features in nucleotide analogs that result in the inhibition of HIV-1 reverse transcriptase, we have evaluated this enzyme with the 5'-triphosphate of the carbocyclic analog of 2'-deoxyguanosine (CdG-TP). CdG-TP was a reasonably potent competitive inhibitor of the incorporation of dGTP into DNA by HIV-1 reverse transcriptase using either a RNA or DNA template (Ki, 1 microM). CdG-TP was a good substrate for HIV-1 reverse transcriptase on both templates, but the DNA chain was poorly extended beyond the incorporation of CdG. These results indicate that substitution of ribose with a cyclopentane ring in nucleotides is not well tolerated by HIV-1 reverse transcriptase.
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PMID:Interference with HIV-1 reverse transcriptase-catalyzed DNA chain elongation by the 5'-triphosphate of the carbocyclic analog of 2'-deoxyguanosine. 128 92

We have examined the RNA-dependent and DNA-dependent polymerase and ribonuclease H catalytic activities of human immunodeficiency virus reverse transcriptase using rapid transient kinetic methods with defined synthetic 25/45-mer DNA/RNA and DNA/DNA primer/templates. The Kd value for interaction of the enzyme with duplex DNA was 4.7 nM, and the value for RNA/DNA heteroduplex was of similar magnitude. A pre-steady state burst of nucleoside triphosphate incorporation was observed for both DNA and RNA templates. Analysis of the dATP concentration dependence of the burst rate provided Kd values for dATP of 4 and 14 microM and maximum rates of single nucleotide incorporation, kpol, of 33 and 74 s-1, for DNA and RNA templates, respectively. Subsequent turnovers were limited by the rate of dissociation of the primer/template from the enzyme at rates of 0.18 and 0.06 s-1 for duplex DNA and RNA/DNA heteroduplex, respectively. Analysis of rates of DNA polymerization and RNA cleavage using the RNA template revealed that the two activities are independent of one another. The polymerization rate (4-70 s-1) was dependent on dATP concentration, whereas the RNA cleavage occurred at a constant rate of 10 s-1 over the 100-fold dATP concentration range (2-200 microM). Examination of the RNA cleavage products resulting from a single turnover indicates that the polymerase and ribonuclease domains of the enzyme are separated by a distance corresponding to 19 bases of RNA/DNA heteroduplex, consistent with the recently published crystal structure (Kohlstaedt, L. A., Wang, J., Friedman, J., Rice, P. A., and Steitz, T. A. (1992) Science 256, 1783-1790). Analysis of the kinetics of processive synthesis suggested that the initial binding of dNTP leads to a faster rate of dissociation of DNA from the enzyme. Further investigation supported a two-step dNTP binding mechanism with the formation of an initial E.DNA.dNTP complex followed by a more stable E'.DNA.dNTP complex. The Kd values for incorporation of incorrect nucleoside triphosphates opposite a DNA template thymidine were 1010 microM for dGTP, 1240 microM for dCTP, and 840 microM for dTTP. The corresponding maximum kpol rates were 4.8 s-1 for dGTP, 0.52 s-1 for dCTP, and 0.41 s-1 for dTTP. These values provide fidelity estimates of 1740 for discrimination against dGTP, 19,700 for dCTP, and 16,900 for dTTP misincorporations at this site.
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PMID:Mechanism and fidelity of HIV reverse transcriptase. 128 79

The in vitro fidelity of reverse transcriptase from human immunodeficiency virus type I (HIV-1 RT) upon copying an RNA template was measured using the phi Xam 16 reversion assay. A phi X174 sequence harboring the amber 16 codon was cloned into a transcription vector. RNA obtained from transcription by bacteriophage T7 RNA polymerase was used as a template for RNA-directed DNA synthesis by HIV-1 RT. An imbalance of dNTP concentrations during the reverse transcription step served to distinguish between errors that arose from the transcription step and errors from reverse transcription. The frequency of dGTP.U mismatches was determined to be 1/360, while dGTP.rA mismatches formed at a rate of 1/4600. These are 20-fold and sevenfold higher, respectively, than the error rates determined for the same sequence with a DNA template. Due to a high background of errors in the RNA template originating from the transcription step only upper limits for the frequency of three other mismatches can be given. The data indicate that the reverse transcription step of the HIV-1 replication cycle contributes significantly to the generation of mutant viruses.
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PMID:Fidelity of human immunodeficiency virus type I reverse transcriptase in copying natural RNA. 137 12

The dipyridodiazepinone derivative 6,11-dihydro-11-cyclopropyl-4-methyldipyrido[2,3-b:2',3'-e]-[1,4] diazepin-6-one (BI-RG-587) selectively inhibits human immunodeficiency virus type 1 (HIV-1) replication by suppressing HIV-1 reverse transcriptase activity. Both RNA- and DNA-dependent polymerase associated activities of this enzyme were found to be inhibited by BI-RG-587 in a pattern dependent on the template used. The lowest IC50 values were obtained using poly(rC)-oligo(dG)12-18 and poly(dA)-oligo(dT)12-18 as template-primer. For the RNA-dependent activity poly(rC)-oligo(dG)12-18 and dGTP appeared to enhance the inhibition of the RNA-dependent enzyme activity by BI-RG-587, with the effect of poly(rC)-oligo(dG)12-18 dominating that of dGTP. Poly(rA)-oligo(dT)10 seemed to decrease the inhibition whereas poly(rU)-oligo(dA)12-18 or poly(rG)-oligo-(dC)12-18 had no effect. dATP, dTTP and dCTP, three nucleotide triphosphates, also had no impact on the inhibition. Differences were observed for the template-dependent action of BI-RG-587 against the DNA-dependent enzyme activity. Both substrates were required to allow the inhibition by BI-RG-587 in the poly(dC)-oligo(dG)12-18 and dGTP reaction, whereas only the template and enzyme interaction seemed to be necessary for the poly(dA)-oligo(dT)12-18 and dTTP reaction. The different behaviors of DNA- and RNA-dependent DNA polymerase activities could indicate either the presence of different active sites for distinct activities or the presence of a unique active site with different configurations depending upon the template used. Also, BI-RG-587 showed a mutually exclusive inhibition when combined with two other classes of HIV-1 RT inhibitors represented by phosphonoformic acid and 3'-azido-3'-dideoxythymidine triphosphate.
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PMID:HIV-1 reverse transcriptase inhibition by a dipyridodiazepinone derivative: BI-RG-587. 137 83

Recently, tetrahydroimidazo-[4,5,1-jk][1,4]-benzodiazepin-2(1H)-one and -thione (TIBO) and 1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)thymine (HEPT) compounds have been shown to be potent, selective, and specific inhibitors of human immunodeficiency virus type 1 (HIV-1) replication in vitro. They interact with the reverse transcriptase of HIV-1 in a way different from that of previously studied reverse transcriptase (RT) inhibitors. We established an endogenous RT assay, starting from intact HIV-1 virions. This assay mimics the reverse transcription process in the HIV-infected cell more closely than RT assays with artificial templates. We investigated the inhibition of endogenous HIV-1 reverse transcription by the TIBO derivative (+)-(S)-4,5,6,7-tetrahydro-5-methyl-6-(3-methyl-2-butenyl)imidazo [4,5,1-jk][1,4]-benzodiazepin-2(1H)-thione (R-82150) in comparison with the HEPT derivative 5-ethyl-1-ethoxymethyl-6-(phenylthio)uracil (E-EPU) and 2',3'-dideoxyguanosine 5'-triphosphate. The kinetics and characteristics of RT inhibition by TIBO in the endogenous RT assay were similar to those found previously for the exogenous RT assay (following addition of exogenous template/primer); thus, RT inhibition by TIBO was specific for HIV-1 and the extent of RT inhibition was dependent on which of the four substrates (dATP, dTTP, dGTP, and dCTP) was present in limited concentrations. Of the three enzymatic activities, RNA-dependent DNA polymerization was preferentially inhibited, and inhibition was not competitive with respect to the natural substrates. HIV-1 RT behaved as an allosteric enzyme, which means that positive cooperativity for binding of the substrate was observed. TIBO behaved as an allosteric inhibitor by causing a concentration-dependent decrease in this cooperativity.
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PMID:Kinetics of inhibition of endogenous human immunodeficiency virus type 1 reverse transcription by 2',3'-dideoxynucleoside 5'-triphosphate, tetrahydroimidazo-[4,5,1-jk][1,4]-benzodiazepin-2(1H)-thion e, and 1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)thymine derivatives. 137 11


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