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)

A new and sensitive assay of reverse transcriptase (RT) activity of retroviruses measures the incorporation of digoxigenin-labelled dUTP in newly synthesized DNA instead of radioactively labelled (3H- or 32P-)dTTP. To avoid difficulties associated with separation of non-incorporated nucleotides from the newly synthesized DNA, biotin-labelled dUTP is added to the reaction mixture in very low concentrations. After reverse transcription, the newly synthesized, doubly labelled DNA is immobilized on streptavidin-coated ELISA wells and evaluated photometrically by binding of peroxidase-conjugated anti-digoxigenin-antibodies (sheep) and subsequent colour development with 2,2'-azino-di[3-ethylbenzthiazolin-sulfonate(6)] (ABTSR) as substrate. For better standardization, it is suggested that RT activity is given in units (one unit of RT is the amount of enzyme incorporating one nanomole of labelled dNTP in 10 min at 37 degrees C into an acid precipitable DNA) rather than in cpm (counts per minute). The method is specific and easy to perform.
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PMID:A new method for measuring reverse transcriptase activity by ELISA. 128 32

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

Mechanisms of the effects of the dTTP analogues 3'-azido-3'-deoxythymidine 5'-triphosphate (AZTTP) and 3'-amino-3'-deoxythymidine 5'-triphosphate (NH2 TTP) upon the HIV-1 reverse transcriptase (RT) are discussed. These compounds block the RT in vitro and do so by different kinetic mechanisms. Infidelity of replication is a hallmark of the HIV-1 RT, and replication errors by the enzyme on RNA and DNA templates are discussed. The enzyme's infidelity has ramifications for inhibition: On the one hand, the propensity to produce mutations enhances the ability of the virus to escape inhibitors whereas on the other hand, the infidelity of the reverse transcriptase may allow the development of imaginative inhibitor strategies.
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PMID:Inhibitors of HIV-1 reverse transcriptase and fidelity of in vitro DNA replication. 128 1

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

Steady-state and pre-steady-state kinetic constants were determined for reverse transcriptase catalyzed incorporation of nucleotides and nucleotide analogues into defined-sequence DNA primed-RNA templates. 3'-Azido-3'-deoxythymidine 5'-triphosphate (AZTTP) was almost as efficient a substrate (kcat/Km) as dTTP for the enzyme. In contrast, the four 2',3'-dideoxynucleoside 5'-triphosphates and 3'-deoxy-2',3'-didehydrothymidine 5'-triphosphate (d4TTP) were 6-30-fold less efficient substrates of the enzyme. The kcat values for all nucleotide analogues were similar, consistent with a kinetic model in which the steady-state rate-limiting step was dissociation of the template-primer from the enzyme [Reardon, J. E., & Miller, W. H. (1990) J. Biol. Chem. 265, 20302-20307]. The pre-steady-state kinetics of single-nucleotide incorporation were consistent with the kinetic model: [formula: see text] where E, TP, and dNTP represent reverse transcriptase, a defined-sequence DNA primed-RNA template, and 2'-deoxynucleoside 5'-triphosphate (or analogue), respectively. The dissociation constant (Kd1) for template-primer binding was 10 nM, and the estimated rate constants for association and dissociation of the enzyme.template-primer complex were 4 x 10(6) M-1 s-1 and 0.04 s-1, respectively. The dissociation constants (Kd2) for dTTP, AZTTP, and 3'-deoxythymidine 5'-triphosphate (ddTTP) were 9, 11, and 4.6 microM, respectively. Thus, the differences in steady-state Km values were not due to differences in binding of the nucleotide analogues to the enzyme. In contrast, the rate-limiting step during single-nucleotide incorporation (kp) was sensitive to the structure of the nucleotide substrate.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Human immunodeficiency virus reverse transcriptase: steady-state and pre-steady-state kinetics of nucleotide incorporation. 137 38

We have previously expressed the cauliflower mosaic virus (CaMV) reverse transcriptase (RTase) gene, the ORFV gene, in yeast in an active form (RTase-Y). An activity gel analysis revealed that the molecular size of RTase-Y as well as an RTase associated with the CaMV particles (RTase-V) is 60 kDa. This size is about 18 kDa smaller than that of the inactive form previously expressed in Escherichia coli (RTase-E) (78 kDa), which corresponds to the coding capacity estimated for the ORFV gene. To investigate the possible involvement of proteolytic processing in the de novo synthesis of CaMV RTase, we constructed a series of deletions from either terminus or both termini of the ORFV coding sequence and expressed them in E. coli. Among the various truncated RTases, those (denoted delta N) that lack N-terminal peptide fragments 143-185 amino acids long were active on the synthetic RNA template-primer, poly(rC)-oligo(dG). Those RTases (denoted delta C) lacking C-terminal peptide fragments 50-102 amino acids long and those lacking both termini (denoted delta NC) were also active on this template. However, only the delta N RTases showed enzyme properties indistinguishable from the RTase-Y in that they transcribed natural RNA into DNA and required either Mg2+ or Mn2+ for their activity. The length of the deletion corresponded approximately to the difference of the molecular weights between RTase-Y and RTase-E. These results suggest that CaMV RTase is translated in an inactive precursor form and then converted to an active form by proteolytic processing during de novo synthesis. We have also demonstrated that C-terminal deletions cause a loss of activity on a natural RNA template accompanied by an alteration in metal ion requirement. The inability to incorporate dTTP accounts for the loss of activity on the natural RNA template. However, the affinities for dTTP and the corresponding template, poly(rA)-oligo(dT), were found to be unaltered.
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PMID:Cauliflower mosaic virus reverse transcriptase. Activation by proteolytic processing and functional alteration by terminal deletion. 137 43

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

The kinetics of copying of poly(A).(dT)n, poly(A).(U)n, poly(dA).(dT)n and poly(A).(dT)9-U by reverse transcriptase of human immunodeficiency virus-1 (HIV-1) has been studied and the binding affinity of the enzyme, for template or primer, determined. Short oligonucleotides and dTTP served as primers in the HIV-1 reverse-transcriptase-dependent DNA synthesis. Km and Vmax were measured as functions of the primer chain length; the logarithm of the values of both Km and Vmax increased linearly up to 10. For longer primers (n = 11 to n = 24) the increase of those values changes very little. The enhanced affinity of the primers, (dT)n or (U)n due to the formation of one complementary pair, A.dT, dA.dT, A.U was estimated as a factor of 2. A specific property of HIV-1 reverse transcriptase compared with other DNA polymerases (procaryotes, eucaryotes, other retroviruses and archaebacteria) was its higher affinity to riboprimers as compared to deoxyriboprimers. Relative initial rates when copying poly(A) or poly(dA) templates using different primers and various conditions were compared; the optimal temperature for the reaction of polymerization with poly(A) or poly(dA) templates and (U)10, (dT)10 or (dT)9-U primers was determined. The maximal activity of the enzyme in the case of poly(A) and decanucleotide primers was found at temperatures between 27-31 degrees C. An increase in the primer length results in the stabilization of the template.primer duplex complexed to the enzyme, thus increasing to more than 40 degrees C the optimal temperature of polymerization. The activation energy (Ea) values of the polymerization reaction for different template.primer complexes were evaluated.
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PMID:Functional analysis of primers and templates in the synthesis of DNA catalyzed by human immunodeficiency virus type 1 reverse transcriptase. 137 4

msDNA-Ec67 is produced in a clinical strain of Escherichia coli and composed of a 67-base single-stranded DNA, which is linked to the 2'-OH group of the 15th rG residue of a 58-base RNA molecule by a 2',5'-phosphodiester linkage (Lampson, B. C., Sun, J., Hsu, M.-Y., Vallejo-Ramirez, J., Inouye, S., and Inouye, M. (1989) Science 243, 1033-1038). The production of msDNA-Ec67 is dependent upon retron-Ec67, which consists of the msr-msd region and the gene for reverse transcriptase (RT). These two elements were separately cloned into plasmids; p67-BHO.6 contained the msr-msd region and pRT-67 contained the RT gene under the lpp-lac promoter-operator. msDNA-Ec67 was produced only when cells were transformed with both plasmids. In addition, msDNA-Ec67 was synthesized in a cell-free system using total RNA prepared from cells harboring plasmid p67-BHO.6 and purified Ec67-RT. Using this cell-free system, the priming reaction, during initiation of DNA synthesis, was demonstrated to be a specific template-directed event; only dTTP was incorporated into a 132-base precursor RNA yielding a 133-base compound. This specific dT addition could be altered to dA or dC by simply substituting the 118th A residue of the putative msr-msd transcript with a T or G residue. The priming reaction was blocked when A was substituted for G at the 15th residue of the precursor RNA transcript, which corresponds to the branched rG residue in msDNA. DNA chain elongation could be terminated by adding ddNTP in the cell-free system, forming a sequence ladder. The DNA sequence determined from this ladder completely agreed with the msDNA sequence. The RT extension reaction was completely blocked when the RNA preparation was treated with RNase A but not when the preparation was treated with DNase. This clearly demonstrates that RNA but not DNA is responsible for the msDNA production. A part of the fully extended cell-free product contained a 13-base RNA strand resistant to RNase A, which is consistent with the previously proposed model. In this model, the 5'-end sequence of the msr-msd transcript (a2; bases 1-13) forms a duplex with the 3'-end sequence (a1) of the same transcript, thus serving as a primer, as well as a template for msDNA synthesis by RT. Our results are inconsistent with a model recently proposed by Lease and Yee (Lease, R. A., and Yee, T. (1991) J. Biol. Chem. 266, 14497-14503).
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PMID:Cell-free synthesis of the branched RNA-linked msDNA from retron-Ec67 of Escherichia coli. 137 31

Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) (EC 2.7.7.49) with a high specific activity has been purified from the overexpressing Escherichia coli strain DH5 alpha [pJS3.7]. Steady-state kinetics of DNA synthesis catalysed by RT were analysed on polyriboadenylate 20-mer of (3'-5')deoxythymidylate [poly(rA).(dT)20] and polyribouridylate 20-mer of (3'-5')-deoxyadenylate [poly(rU).(dA)20] homopolymeric template-primers. Km values of 40 and 140 nM (3'-OH ends) and kcat values of 4 and 0.14 sec-1 were determined for the two different substrates. Oligonucleotide primers (dA)20 and (dT)20 were elongated in a terminal transferase-catalysed reaction (EC 2.7.7.31) with ddATP, 3'-dATP (cordycepin), 2',3'-epoxy-ATP and arabino-ATP; and ddTTP, 3'-azido-TTP, 3'-dUTP, 3'-F-dTTP and rUTP, respectively. The resulting oligonucleotides were hybridized to their complementary templates and the inhibitory potential of these compounds towards DNA synthesis started from unchanged primers was measured. Oligonucleotides with unextendable 3'-groups were shown to act as strong inhibitors of DNA synthesis catalysed by HIV-1 RT. In particular, poly(rA).(dT)20-[ddTMP] and poly(rU).(dA)20-[3'-dAMP] were potent competitive inhibitors, displaying Ki values of about 6 and 12 nM, respectively. Also 3'-azido-, and 3'-fluoro-terminated oligonucleotides showed competitive inhibition with inhibition constants in the range of 20-35 nM. In contrast, 2',3'-epoxy-terminated (dA)21 displayed a mixed-type inhibition with a Ki value of 67 nM. Arabino-terminated (dA)21 was found to be an uncompetitive inhibitor of HIV-1 RT with an inhibition constant of 318 nM. Arabino-terminated primers did not act as strict chain terminators because they could be elongated by HIV-1 RT. This study provides information on the structure-activity relationship of modified 3'-termini of primer molecules which might be exploited as inhibitors of HIV in the future.
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PMID:Inhibition of human immunodeficiency virus type 1 reverse transcriptase by 3'-blocked oligonucleotide primers. 137 38


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