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)

beta-L-3'-Deoxythymidine 5'-triphosphate (L-ddTTP) and beta-L-3'-deoxy-2',3'-didehydrothymidine 5'-triphosphate (L-d4TTP) were substrates for human immunodeficiency virus reverse transcriptase, Escherichia coli DNA polymerase I (Klenow), and Sequenase (modified T7 DNA polymerase). The beta-D- and beta-L-enantiomers of 5-methyluridine 5'-triphosphate (rTTP) were inhibitors but not substrates of reverse transcriptase. The steady-state Km values for L-ddTTP and L-d4TTP, with all three enzymes, were 12-70-fold larger than the Km values for the corresponding D-enantiomers. The Km value of reverse transcriptase for L-ddTTP was 50-fold larger than that for D-ddTTP because the Kd for L-ddTTP was 5-fold larger than that for D-ddTTP, and the first-order rate constant for incorporation of L-ddTMP into the template-primer was 10% that of the D-enantiomer. The D- and L-enantiomers had kcat values with reverse transcriptase and Sequenase that were similar to kcat for the natural substrate, thymidine 5'-triphosphate (dTTP). Thus, the rate determining step appeared to be dissociation of the enzyme-chain-terminated template-primer complex. In contrast, kcat values for the L-enantiomers with Klenow were only 0.1% that of dTTP, and the kcat values for the D-enantiomers were 15% the kcat for dTTP. The reduced kcat values were due to a change in rate determining step from dissociation of the Klenow-chain-terminated template-primer complex to an earlier step in the reaction mechanism, presumably catalysis. Thus, these DNA polymerases did not stereospecifically recognize D-nucleoside 5'-triphosphate analogs as substrates.
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PMID:Beta-L-thymidine 5'-triphosphate analogs as DNA polymerase substrates. 128 Nov 53

Activities of the hepadnavirus polymerases are known to include those of DNA polymerase, reverse transcriptase and RNase H. To date, it has been difficult or impossible to clone and express the product as an active enzyme. In this study, full length capped RNA encoding Duck Hepatitis B Virus (DHBV) polymerase was produced by in vitro transcription from a T7 promoter. The RNA was translated in a rabbit reticulocyte lysate system and produced an 35S-Methionine labelled 79 Kd band on SDS-polyacrylamide gel electrophoresis. The translation product showed DNA polymerase and reverse transcriptase activities on exogenous templates (respectively) of DNA or RNA with random DNA hexamer primers. The same RNA transcripts were also microinjected into Xenopus oocytes, but appeared to be toxic and gave no detectable translation product. Production of hepadnavirus polymerase by in vitro transcription/translation may provide a useful tool for structure/function and pharmacological studies on this important group of polymerases.
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PMID:Duck hepatitis B virus polymerase produced by in vitro transcription and translation possesses DNA polymerase and reverse transcriptase activities. 128 90

The method of DHBV replication complexes (RCs) purification was modified. In order to screen anti-HBV drugs from Chinese medicinal herbs, the inhibitory effects of the extracts of 14 Chinese recipes and herbs, 30 compounds isolated from Chinese herbs on DHBV DNA polymerase (DNAP) and reverse transcriptase (RT) have been studied. The results showed that extracts of Xiao Chai Hu Tang (small Bupleurum decoction) inhibited DHBV DNAP and RT in less extent. Of the 7 herbs, the components of Xiao Chai Hu Tang, the extracts of S. baicalensis and P. ternata potently inhibited DHBV RT, their concentration of reducing enzyme activity by 50% (IC50) was 1.25 and 1.6 mg/ml respectively. Furthermore, it has been proved that S. baicalensis inhibited DHBV DNA replication in ducklings. It also was found the extract of P. cuspidatum inhibited DHBV RT with IC50 of 1.76 mg/ml. Nine of thirty isolated compounds inhibited both DHBV DNAP and RT in less extent under high concentration, while other did not.
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PMID:[Uses of duck hepatitis B virus polymerase and reverse transcriptase in the evaluation of anti-hepatitis B virus drugs]. 128 99

Catechin derivatives including (-)-epicatechin gallate (ECG), (-)-epigallocatechin gallate (EGCG), (-)-epigallocatechin (EGC) and green tea extract (GTE) were found to inhibit the activities of cloned human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT), duck hepatitis B virus replication complexes reverse transcriptase (DHBV RCs RT), herpes simplex virus 1 DNA polymerase (HSV-1 DNAP) and cow thymus DNA polymerase alpha (CT DNAP alpha). EGCG and ECG were shown to be very potent inhibitors of HIV-1 RT. According to the IC50 values for HIV-1 RT, these compounds can be ordered as EGCG 0.0066 mumol/L > ECG 0.084 mumol/L > GTE 0.1 microgram/ml > EGC 7.2 mumol/L. DHBV RCs RT was the least sensitive to these compounds. Kinetic study showed that EGCG exerts a mixed inhibition with respect to external template inducer poly (rA).oligo (dT) 12-18 and a noncompetitive inhibition with respect to substrate dTTP for HIV-1 RT. Bovine serum albumin significantly reduced the inhibitory effects of catechin analogues and GTE on HIV-1 RT. In tissue culture GTE inhibited the cytopathic effect of coxsackie B3 virus, but did not inhibit the cytopathic effects of HSV-1, HSV-2, influenza A or influenza B viruses.
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PMID:[The inhibitory effects of catechin derivatives on the activities of human immunodeficiency virus reverse transcriptase and DNA polymerases]. 128 89

The inhibitor sensitivity and functional domains of recombinant encephalomyocarditis (EMC) virus RNA-dependent RNA polymerase (3Dpol) have been extensively analyzed. The inhibitor profiles of EMC virus 3Dpol and Escherichia coli DNA-dependent RNA polymerase are distinct, and experiments with substrate analogs indicate that EMC virus 3Dpol lacks reverse transcriptase activity. Twenty amino acid substitutions were engineered in EMC virus 3Dpol based on sequence alignments of viral RNA-dependent RNA polymerases that identified conserved amino acid residues within motifs. Ten out of 17 conservative substitutions within the four most conserved motifs reduced the RNA polymerase activity of the mutants to 0-6% of the activity of the wild-type enzyme, demonstrating the importance of these amino acids in the structure and/or function of EMC virus 3Dpol. Remarkably, 5 of the 10 mutations in EMC virus 3Dpol which had the most drastic effect on its RNA polymerase activity (D240E, S293T, N302Q, G332A, and D333E) were found to correspond to active site residues in E. coli DNA-dependent DNA polymerase I (Klenow). Our results reveal that a basic structural and functional framework is conserved in the most distantly related classes of nucleic acid polymerases and demonstrate the validity of modeling the active site of an RNA-dependent RNA polymerase on the known structure of a DNA polymerase.
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PMID:Point mutations which drastically affect the polymerization activity of encephalomyocarditis virus RNA-dependent RNA polymerase correspond to the active site of Escherichia coli DNA polymerase I. 131 53

A synthetic RNA oligonucleotide (15-mer) corresponding to the 3' end of the lysine tRNA primer was hybridized to single-stranded DNA containing the human immunodeficiency virus type 1 (HIV-1) primer-binding site and extended with a DNA polymerase. The resulting structures were used to study primer removal by the RNase H activity of HIV-1 reverse transcriptase. The initial cleavage event removes the RNA primer as a 14-mer and leaves a single ribonucleotide A residue bound to the 5' end of the DNA strand. This result explains the observation by several groups that HIV-1 circle junctions contain 4 bp that are not present in the integrated provirus instead of the predicted 3 bp. Subsequent cleavage events occur at other sites internal to the RNA molecule, and the ribonucleotide A residue on the end of the DNA strand is ultimately removed. Therefore, the biologically relevant cleavage that produces the 14-mer reflects the kinetics of the reaction as well as a specificity for nucleic acid sequence. When the RNA oligonucleotide alone was hybridized to the primer-binding site and tested as a substrate for HIV-1 RNase H, the cleavage pattern near the 3' end of the RNA was altered.
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PMID:Incomplete removal of the RNA primer for minus-strand DNA synthesis by human immunodeficiency virus type 1 reverse transcriptase. 137 87

We have studied a mutant Moloney murine leukemia virus with a deletion in reverse transcriptase (RT) which is predicted to make its RNase H domain resemble structurally that of human immunodeficiency virus RT. This deletion was based on improved RNase H homology alignments made possible by the recently solved three-dimensional structure for Escherichia coli RNase H. This mutant Moloney murine leukemia virus RT was fully active in the oligo(dT)-poly(rA) DNA polymerase assay and retained nearly all of wild-type RT's RNase H activity in an in situ RNase H gel assay. However, proviruses reconstructed to include this deletion were noninfectious. Minus-strand strong-stop DNA was made by the deletion mutant, but the amount of minus-strand translocation was intermediate to the very low level measured with RNase H-null virions and the high level seen with wild-type RT. The average length of translocated minus-strand DNA was shorter for the deletion mutant than for wild type, suggesting that mutations in the RNase H domain of RT also affect DNA polymerase activity.
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PMID:Defects in Moloney murine leukemia virus replication caused by a reverse transcriptase mutation modeled on the structure of Escherichia coli RNase H. 137 May 51

We investigate the enzymatic basis for the inefficient extension of single base mismatches by DNA polymerase compared with the extension of correct base pairs. Inefficient mismatch extension could result from either a reduced binding of the enzyme to mispaired versus correctly paired DNA template-primer termini, or from a lowered intrinsic rate of extension of mispairs by a bound enzyme, or from a combination of both factors. Avian myeloblastosis reverse transcriptase is used to measure the affinities (equilibrium dissociation constants) for the four matched and twelve mismatched base pair configurations situated at a primer 3'-terminus. The binding affinities are analyzed by two different assays employing polyacrylamide gels. The first assay uses steady-state kinetics to measure the efficiency of elongating correct and incorrect base pairs and to evaluate the enzyme's dissociation constants for matched and mismatched termini. The estimated KD values obtained in the steady-state analysis fall within a range of approximately 0.1-20 nM. The efficiencies of extending two of the mispairs, G.G and C.C, are too low to allow a determination of KD by the kinetics method. The second assay uses equilibrium binding to measure the ratio of polymerase bound to matched compared with mismatched termini, KDright/KDwrong. The affinity ratios, including values for G.G and C.C mispairs, are in the range of about 0.4-4.2. While around 1 order of magnitude difference is observed in the relative binding affinities of the polymerase for matched and mismatched primer termini, the relative extension efficiencies vary over more than 5 orders of magnitude. Therefore, it appears that inefficient mismatch extension is caused primarily by a kinetic block inhibiting elongation from mispaired primer 3'-termini rather than to a difference in binding.
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PMID:Base mispair extension kinetics. Binding of avian myeloblastosis reverse transcriptase to matched and mismatched base pair termini. 137 Aug 28

Early events in the retroviral replication cycle include the conversion of viral genomic RNA into linear double-stranded DNA. This process is mediated by the reverse transcriptase (RT), a multifunctional enzyme that possesses RNA-dependent DNA polymerase, DNA-dependent DNA polymerase, and RNase H activities. In the course of studies of a recombinant RT of human immunodeficiency virus type 1 (HIV-1), we observed an additional, unexpected activity of the enzyme. The purified RT catalyzes a specific cleavage in HIV-1 RNA hybridized to tRNALys, the primer for HIV-1 reverse transcription. The cleavage at the primer binding site (PBS) of HIV RNA is dependent on the double-stranded structure of the HIV RNA-tRNALys complex. This RNase activity appears to be distinct from the RNase H activity of HIV-1 RT, as the substrate specificity and the products of the two activities are different. Moreover, Escherichia coli RNase H and avian myeloblastosis virus RT are unable to cleave the HIV RNA-tRNALys complex. We refer to this unusual activity as RNase D. Two lines of evidence indicate that the specific RNase D activity is an integral part of recombinant HIV RT. The specific RNase D activity comigrates with the other RT activities, DNA polymerase, and RNase H upon filtration on a Superose 6 gel column or chromatography on a phosphocellulose column. Moreover, three recombinant HIV-1 RT preparations expressed and purified in different laboratories by various procedures exhibit RNase D activity. Sequence analysis indicated that RNase D activity cleaves the substrate HIV-1 RNA-tRNALys at two distinct sites within the PBS sequence 5'-UGGCGCCCGA decreases ACAG decreases GGAC-3'. The sequence specificity of RNase D activity suggests that it might be involved in two stages during the reverse transcription process: displacement of the PBS to enable copying of tRNALys sequences into plus-strand DNA or to facilitate the second template switch, which was postulated to occur at the PBS sequence.
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PMID:Double-stranded RNA-dependent RNase activity associated with human immunodeficiency virus type 1 reverse transcriptase. 137 Oct 14

Foscarnet is a pyrophosphate analogue with activity against herpesviruses, human immunodeficiency virus (HIV), and other RNA and DNA viruses. Foscarnet and its analogues achieve their antiviral effects via inhibition of viral polymerases, with such inhibition not being dependent on activation or phosphorylation of the compounds by viral or cellular proteins. Current evidence indicates that foscarnet interferes with exchange of pyrophosphate from deoxynucleoside triphosphate during viral replication by binding to a site on the herpesvirus DNA polymerase or HIV reverse transcriptase. Reviewed herein are basic findings regarding the mechanism of action and antiviral activity of foscarnet and the related compound phosphonoacetic acid (PAA), as well as findings regarding potential mechanisms of viral resistance and interactions with other antiviral agents.
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PMID:Mechanism of action of foscarnet against viral polymerases. 137 Oct 38


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