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)

Novobiocin inhibits animal DNA polymerase alpha and avian reverse transcriptase activities when these enzymes are assayed in vitro with activated DNA as template. Under the same conditions DNA polymerase beta and gamma are much less inhibited. DNA polymerase alpha and reverse transcriptase are inhibited by different mechanisms: in the case of the retroviral enzyme the effect of novobiocin is not overcome by dilution of the drug, while in the case of polymerase alpha the inhibition disappeared after novobiocin dilution. The inhibition of polymerase alpha by novobiocin is non-competitive with respect to the TTP precursor or activated DNA. The irreversible inactivation of reverse transcriptase by novobiocin leads to the loss of the enzyme affinity for primer tRNATrp. Moreover, novobiocin inhibits the partial unwinding of the 3' end of tRNATrp by reverse transcriptase.
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PMID:The in vitro inhibition of DNA polymerase alpha and avian reverse transcriptase by novobiocin. 620 65

Enzymatic incorporation of 2',3'-dideoxynucleotides into DNA results in chain termination. We report that 3'-esterified 2'-deoxynucleoside 5'-triphosphates (dNTPs) are false chain-terminator substrates since DNA polymerases, including human immunodeficiency virus reverse transcriptase, can incorporate them into DNA and, subsequently, use this new 3' end to insert the next correctly paired dNTP. Likewise, a DNA substrate with a primer chemically esterified at the 3' position can be extended efficiently upon incubation with dNTPs and T7 DNA polymerase lacking 3'-to-5' exonuclease activity. This enzyme is also able to use dTTP-bearing reporter groups in the 3' position conjugated through amide or thiourea bonds and cleave them to restore a DNA chain terminated by an amino group at the 3' end. Hence, a number of DNA polymerases exhibit wide catalytic versatility at the 3' end of the nascent DNA strand. As part of the polymerization mechanism, these capabilities extend the number of enzymatic activities associated with these enzymes and also the study of interactions between DNA polymerases and nucleotide analogues.
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PMID:Catalytic editing properties of DNA polymerases. 747 98

Inhibitory effects of 2'-deoxy-L-thymidine 5'-triphosphate (L-dTTP) which is an enantiomer of the natural substrate (D-dTTP) on the activity of mammalian DNA polymerases, Escherichia coli DNA polymerase I and HIV-1 reverse transcriptase were examined. Interestingly, L-dTTP showed remarkable inhibitory effect on HIV-1 reverse transcriptase in competitive fashion with respect to the substrate dTTP. In contrast, eukaryotic cell nuclear DNA polymerases alpha and beta were not or slightly inhibited by L-dTTP.
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PMID:Inhibitory effects of 2'-deoxy-L-thymidine 5'-triphosphate on human immunodeficiency virus reverse transcriptase and eukaryotic DNA polymerases. 750 40

Inhibitory effects of 2'-deoxy-L-thymidine 5'-triphosphate (L-dTTP), the enantiomer of the natural substrate D-dTTP, on the activity of mammalian DNA polymerases alpha, beta and gamma, Escherichia coli DNA polymerase I and human immunodeficiency virus 1 (HIV-1) reverse transcriptase were examined. When poly(rA)n-oligo(dT)12-18 was used as the template-primer, L-dTTP showed remarkable inhibitory effect on HIV-1 reverse transcriptase in competitive fashion with respect to the substrate dTTP. In contrast, L-dTTP did not inhibit DNA polymerases alpha and was slightly inhibitory to DNA polymerase beta. These results suggest that the nuclear DNA polymerases alpha and beta showed high specificity for the substrate with the natural configuration of the sugar moiety, D-dTTP, exhibiting little or no ability to recognize L-dTTP, whereas HIV-1 reverse transcriptase essentially lacked the ability to differentiate the D- and L-sugar moieties.
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PMID:Chiral discrimination of enantiomeric 2'-deoxythymidine 5'-triphosphate by HIV-1 reverse transcriptase and eukaryotic DNA polymerases. 751 92

Fifty-seven Thai herbs and spices were examined for their retroviral reverse transcriptase inhibitory activity. All herbs and spices were extracted with hot-water and methanol. Reverse transcriptase inhibitory activity of the extracts was determined by using Moloney Murine Leukemia Virus reverse transcriptase (M-MuLV-RT) reacted with 3H-dTTP and radioactivity measured with a scintillation counter. Eighty-one per cent (46/57) of hot-water extracts and 54% (31/57) of methanol extracts showed inhibitory activities. At a concentration of 125 micrograms/ml, 13% (6/46) of hot-water extracts, namely Eugenia caryophyllus Bullock et Harrison, Phyllanthus urinaria Linn., Terminalia belerica Roxb., Nelumbo nucifera Gaertn., Psidium guajava Linn. and Lawsonia inermis Linn., had a relative inhibitory ratio (IR) over 50%. They showed ratios of 100%, 91%, 75%, 74%, 61% and 60%, respectively. For methanol extracts, only 10% (3/31) had IR values over 50%. They were T. belerica, E. caryophyllus and N. nucifera which exhibited IR values of 83%, 54% and 54%, respectively.
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PMID:Retroviral reverse transcriptase inhibitory activity in Thai herbs and spices: screening with Moloney murine leukemia viral enzyme. 752 65

The finding of G-->A hypermutated retroviral genomes in which up to 40% of guanines may be substituted by adenines was proposed to result from the depletion of the intracellular dCTP concentration and suggested a means to hypermutagenize nucleic acids. Using a RNA/reverse transcriptase ratio of approximately 1:30, comparable to that within the retroviral replication complex, G-->A hypermutants were produced in a simple in vitro reaction using highly biased dNTP concentrations--i.e., a low ratio of [dCTP]/[dTTP]. Up to 38% of G residues could be substituted, the proportion being inversely proportional to the concentration of dCTP. As G-->A hypermutation resulted from elongation beyond multiple rG.dT mismatches, U-->C hypermutants resulting from multiple rU.dG mismatches were sought, and found, during cDNA synthesis using low [dATP] and high [dGTP]. Mixed G-->A and U-->C hypermutants could also be produced under conditions of low [dCTP] plus low [dATP] and high [dTTP] plus high [dGTP]. Hypermutagenesis should allow jumping through, and subsequent exploration of, sequence space to a greater degree than heretofore and, in conjunction with genetic screening, might be of use in the search of proteins or ribozymes with novel or enhanced properties.
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PMID:Hypermutagenesis of RNA using human immunodeficiency virus type 1 reverse transcriptase and biased dNTP concentrations. 752 43

6-Chloro-(4S)-cyclopropyl-3,4-dihydro-4-((2-pyridyl)-ethynyl)quinazol in- 2(1H)-one (L-738,372) is representative of a novel structural class of nonnucleoside inhibitors of human immunodeficiency virus, strain 1 (HIV-1), reverse transcriptase (RT), the quinazolinones. L-738,372 is a reversible inhibitor of HIV-1 RT and is noncompetitive against dTTP with a Ki of 140 nM with poly(rA).oligo(dT) as primer-template. Mixed noncompetitive inhibition by L-738,372 was observed against poly(rC).oligo(dG) as primer-template. This quinazolinone binds to RT at a site that overlaps the binding site of other nonnucleoside inhibitors as evidenced by the ability of L-738,372 to displace bound radiolabeled L-696,229, a member of the pyridinone class of inhibitors of HIV-1 RT, from complexes of RT and primer-template. Inhibition by L-738,372 shows slow binding characteristics in reactions with all of the primer-templates employed. Synergistic inhibition of RT activity was evident in combinations of L-738,372 and any of the nucleoside analogs, azidothymidine triphosphate, dideoxyinosine triphosphate, or dideoxycytosine triphosphate. The azidothymidine-resistant form of RT (D67N, K70R, T215Y, K219Q) is inhibited by L-738,372 with 2-3-fold more potency than is the wild-type RT. Comparison of inhibition by L-738,372 with inhibition by pyridinone inhibitors reveals differences in synergistic inhibition with nucleoside analogs and in the rates of binding of the inhibitors.
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PMID:Inhibition of HIV-1 reverse transcriptase by a quinazolinone and comparison with inhibition by pyridinones. Differences in the rates of inhibitor binding and in synergistic inhibition with nucleoside analogs. 752 14

When the single-stranded RNA genome of HIV-1 is copied into double-stranded DNA, the viral enzyme reverse transcriptase (RT) catalyzes the addition of approximately 20,000 nucleotides; however, the precise mechanism of nucleotide addition is unknown. In this study, we attempt to integrate the genetic data and biochemical mechanism of DNA polymerization with the structure of HIV-1 RT complexed with a dsDNA template-primer. The first step of polymerization involves the physical association of a polymerase with its nucleic acid substrate. A comparison of the structures of HIV-1 RT in the presence and absence of DNA indicates that the tip of the p66 thumb moves approximately 30 A upon DNA binding. This conformational change permits numerous interactions between residues of alpha-helices H and I in the thumb subdomain and the DNA. Measurements of DNA binding affinity for nucleic acids with double-stranded DNAs that have an increasing number of bases in the template overhang and molecular modeling suggest that portions of beta 3 and beta 4 within the fingers subdomain bind single-stranded regions of the template. Measurements of nucleotide incorporation efficiency (kcat/Km) show that the binding and incorporation of the next complementary nucleotide are not dependent on the length of the template overhang. Molecular modeling of an incoming nucleotide triphosphate (dTTP), based in part on the position of mercury atoms in a RT/DNA/Hg-UTP/Fab structure, suggests that portions of secondary structural elements alpha C-beta 6, alpha E, beta 11b, and beta 9-beta 10 determine the topology of the dNTP-binding site. These results also suggest that nucleotide incorporation is accompanied by a protein conformational change that positions the dNTP for nucleophilic attack. Nucleophilic attack by the oxygen atom of the 3'-OH group of the primer strand could be metal-mediated, and Asp185 may be directly involved in stabilizing the transition state. The translocation step may be characterized by rotational as well as translational motions of HIV-1 RT relative to the DNA double helix. Some of the energy required for translocation could be provided by dNTP hydrolysis and could be coupled with conformational changes within the nucleic acid. A structural comparison of HIV-1 RT, Klenow fragment, and T7 RNA polymerase identified regions within T7 RNA polymerase which are not present in the other two polymerases that might help this polymerase to remain bound with nucleic acids and contribute to the ability of the T7 RNA polymerase to polymerize processively.
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PMID:Insights into DNA polymerization mechanisms from structure and function analysis of HIV-1 reverse transcriptase. 753 90

We describe a rapid fluorometric assay for reverse transcriptase (RT) activity. After RT is incubated in the presence of poly(A).oligo(dT) and dTTP for up to 1 h, the reaction is stopped with EDTA and aliquots are added to cuvettes containing 4',6-diamidino-2-phenylindole (DAPI). DAPI fluorescence, which is increased upon binding the RNA.DNA heteroduplex, is measured after 30 min and is linearly dependent on the enzymatic reaction time and the amount of active RT added to the enzyme assay. The increased fluorescence correlates well with the incorporation of [alpha-32P]dTTP into DNA (r2 = 0.986). However, similar assays with the Klenow fragment using poly(dA).oligo(dT) did not result in increased fluorescence under conditions wherein incorporation of [alpha-32P]dTTP into DNA was documented. Thus, the poly(A).poly(dT) [RNA.DNA] heteroduplex must differ from the poly(dA).poly(dT) [DNA.DNA] duplex in a manner that allows for a perturbation of DAPI fluorescence. The relative specific activities of RT in crude preparations measured with the fluorometric assay were comparable to conventional isotopic enzyme assays as were determinations for the type of inhibition and the kinetic constants of purified RT with inhibitors such as zidovudine 5'-triphosphate, nevirapine, and oltipraz.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Fluorometric measurement of reverse transcriptase activity with 4',6-diamidino-2-phenylindole. 753 86

Preparations of Rous sarcoma virus reverse transcriptase isolated from a culture of E. coli HB101 (pMF14) and purified to homogeneity were used to study the steady state kinetics of DNA polymerization and inhibition of DNA-polymerase activity. DNA synthesis was examined using a system of poly(rA) as template, oligo(dT) as primer and dTTP as nucleotide substrate. Kinetic constants for steady state conditions were determined. The substrate initial velocity patterns point to an ordered mechanism which results in the formation of a ternary complex, in which the template-primer is the first to bind to the enzyme. Inhibition of the DNA-polymerase activity of the enzyme by various inhibitors was studied. Analysis of final products of the DNA-polymerase reaction revealed the presence of distribution syntheses of the DNA chain by the alpha alpha-subunit form of the enzyme.
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PMID:[Recombinant reverse transcriptase from Rous sarcoma virus. Kinetics and inhibition of DNA polymerase activity]. 754 30


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