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:2.7.7.48 (transcriptase)
9,479 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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

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

3'-Azido-3'-deoxythymidine triphosphate (erythro) and its threo isomer were synthesized and investigated for their inhibition of HIV reverse transcriptase from virus isolate U 937/HTLV-III. The erythro isomer was a competitive inhibitor of the reaction directed by (rA)n(dT)12-18 and the Ki value was 0.0022 microM. The threo isomer was at least 100-fold less active. The inhibition was specific for dTMP incorporation, and dGMP incorporation using (rC)n(dG)12-18 as template/primer was not affected. The Km value for dTTP varied between 0.7 microM and 1.7 microM. Reverse transcriptase from nine HIV isolates were tested for inhibition by the erythro isomer and only slight differences in sensitivity were observed.
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PMID:Inhibition of the reverse transcriptase from HIV by 3'-azido-3'-deoxythymidine triphosphate and its threo analogue. 244 Mar 80

3-Methylthymine was synthesized into DNA copolymers and deoxynucleoside triphosphate to study its effect on DNA synthesis by the Klenow fragment of Escherichia coli polymerase I and avian myeloblastosis virus reverse transcriptase. Both polymerases were greatly inhibited by template 3-methylthymine. In response to 3-methylthymine, misincorporation of dTTP increased slightly, but occurred only at low levels consistent with spontaneous misincorporation in vitro. Surprisingly, template 3-methylthymine resulted in a striking decrease in background misincorporation, relative to normal incorporation by the Klenow fragment, of dGTP and, to a lesser extent, of dATP and dCTP. The incorporation of 3-methyl-dTTP into DNA was studied using DNA sequencing technology. The Klenow fragment failed to incorporate 3-methyl-dTTP even at 1 mM. Reverse transcriptase incorporated 3-methyl-dTTP opposite adenine, cytosine, and thymine, but at only about 1/40,000th the efficiency of complementary deoxynucleoside triphosphate incorporation. Furthermore, synthesis generally stalled at sites of 3-methyl-thymine incorporation. From these results, we conclude that damage at the central hydrogen-bonding position of thymine abolishes its base-pairing capabilities during DNA synthesis.
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PMID:DNA damage at thymine N-3 abolishes base-pairing capacity during DNA synthesis. 244 69

Reverse transcriptase was purified from human immunodeficiency virus (HIV). It utilized the artificial primer-template poly(rA)-oligo(dT)12-18 more efficiently than activated calf thymus DNA, poly(rI)-oligo(dC)12-18, poly(rC)-oligo(dG)12-18, or poly(rCm)-oligo(dG)12-18. Maximum activity was observed at pH 7.0 to 7.6 in the presence of 5 mM MgCl2 and 100 mM KCl. 3'-Azido-3'-deoxythymidine triphosphate competed with dTTP for binding to HIV reverse transcriptase. Different kinetic constants were obtained with different primer-templates. Km and Ki values of 2.8 and 0.04 microM, respectively, were obtained with poly(rA)-oligo(dT)12-18. The corresponding values were 1.2 and 0.3 microM, respectively, with activated calf thymus DNA and 0.3 and 0.01 microM, respectively, with extracted virus and native template. Inhibition of the host cell DNA polymerases alpha and beta was considerably weaker. The Km and Ki values obtained with activated calf thymus DNA as the primer-template were 2.4 and 230 microM, respectively, for DNA polymerase alpha and 6.0 and 73 microM, respectively, for DNA polymerase beta. 3'-Azido-3'-deoxythymidine triphosphate could also serve as an alternate substrate for HIV reverse transcriptase. The resulting incorporation of 3'-azido-3'-deoxythymidine triphosphate into poly(rA)-oligo(dT)12-18 caused chain termination and premature deceleration of the reaction. The terminated primer could not be elongated when incubated with dTTP and HIV reverse transcriptase.
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PMID:3'-Azido-3'-deoxythymidine triphosphate as an inhibitor and substrate of purified human immunodeficiency virus reverse transcriptase. 244 66

We have analyzed the kinetics of DNA synthesis catalyzed by reverse transcriptase from human immunodeficiency virus 1 (HIV-1). Reverse transcriptase, overproduced in Escherichia coli and purified to homogeneity, has polymerase and RNase H activity. Reverse transcriptase forms a stable complex with poly(rA).oligo(dT) primer-templates in the absence of Mg2+ and dTTP with an equilibrium dissociation constant of 3 nM. Synthesis from these preformed complexes can be initiated, and restricted to a single processive cycle, by the simultaneous addition of Mg2+, dTTP, and excess competitor RNA. Preformed complexes decay with a maximal half-life of 2-3 min. Synthesis on poly(rA) templates is processive with an incorporation rate of 10-15 nucleotides/s at 37 degrees C. Processivity varies widely with the template used, increasing from a few to greater than 300 nucleotides in the order: poly(dA) less than double-stranded DNA less than single-stranded DNA less than single-stranded RNA less than poly(rA). On double-stranded DNA reverse transcriptase catalyzes limited strand-displacement synthesis of up to 50 nucleotides. On RNA-DNA hybrids significant DNA synthesis is observed only after degradation of the RNA strand by the RNase H activity of reverse transcriptase. Intermolecular strand switching occurs with poly(rA) templates. At low ionic strength reverse transcriptase can use multiple templates with a single primer, leading to products of greater than template length. Reverse transcriptase and primer do not have to dissociate during the exchange of template strands, thus allowing processive DNA synthesis across template borders.
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PMID:Human immunodeficiency virus 1 reverse transcriptase. Template binding, processivity, strand displacement synthesis, and template switching. 246 38

Reverse transcriptase from the simian immunodeficiency virus (SIV) was found to have kinetic behavior similar to that of enzyme from the human immunodeficiency virus (HIV). Michaelis constants for the substrates TTP and dGTP and inhibition constants for the inhibitors 3'-azido-3'-deoxythymidine 5'-triphosphate, 2',3'-dideoxythymidine 5'-triphosphate, and 2'-3'-dideoxyguanosine 5'-triphosphate were obtained for SIV reverse transcriptase and were found to be similar to the corresponding values for HIV reverse transcriptase. Thus, the interaction of SIV reverse transcriptase with nucleotide analogs appears to be indistinguishable from that of the HIV enzyme, suggesting that SIV/simian acquired immunodeficiency syndrome (SAIDS) is a potentially good model of AIDS.
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PMID:Kinetics and inhibition of reverse transcriptase from human and simian immunodeficiency viruses. 246 88

Two human strains (AD-169 and C87) and one simian strain (GR2757) of cytomegalovirus (CMV) have been purified from the extracellular fluids of virus-infected cultures by sedimentation through a sucrose gradient followed by brief centrifugation in a preformed gradient of CsCl. Enveloped virus particles were located in the density region, 1.219 g/cm(3), and nucleocapsids at 1.263 g/cm(3). Purified viral DNA, both human and simian, sedimented in the region of 55S in neutral sucrose gradients with herpes simplex type I DNA as a marker; the molecular weight of the CMV DNA was estimated as approximately 10(8). The density of the viral DNA determined by analytical ultracentrifugation was 1.716 g/cm(3) for the human strains and 1.710 g/cm(3) for the simian strain. Tritiated viral complementary RNA synthesized in vitro with Escherichia coli transcriptase has been used for detection and localization of viral genome in membrane hybridization and in situ cytohybridization. Newly synthesized viral DNA appeared 24 h after infection and localized at two acrocentric areas; later the viral DNA distributed in a band resembling intranuclear inclusions 48 to 70 h after infection. Total DNA synthesis began to increase 24 h after infection and reached its peak at 70 h; RNA synthesis increased at 13 h, and reached its peak at 24 to 33 h. The viral DNA was also labeled with (3)H-TTP by repair-synthesis in vitro with Kornberg's enzyme in order to analyze the purity of the DNA and for detection of viral DNA by DNA-DNA reassociation kinetics.
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PMID:Human cytomegalovirus. I. Purification and characterization of viral DNA. 412 79

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

We have selected and plaque purified a mutant of feline immunodeficiency virus (FIV) that is resistant to 2',3'-dideoxycytidine (ddC). This mutant was selected in cultured cells in the continuous presence of 25 microM ddC. The mutant, designated DCR-5c, was fourfold resistant to ddC, threefold resistant to 2',3'-dideoxyinosine, and more than fourfold resistant to phosphonoformic acid. DCR-5c displayed little or no resistance to (-)-beta-2',3'-dideoxy-3'-thiacytidine, 3'-azido-3'-deoxythymidine, or 9-(2-phosphonylmethoxyethyl) adenine. Reverse transcriptase purified from DCR-5c was less susceptible to inhibition by ddCTP, phosphonoformic acid, ddATP, or azido-dTTP than the wild-type FIV reverse transcriptase. Sequence analysis of DCR-5c revealed a single base change (G to C at nucleotide 2342) in the reverse transcriptase-encoding region of FIV. This mutation results in substitution of His for Asp at codon 3 of FIV reverse transcriptase. The role of this mutation in ddC resistance was confirmed by site-directed mutagenesis.
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PMID:Selection and characterization of a mutant of feline immunodeficiency virus resistant to 2',3'-dideoxycytidine. 884 58


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