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:2.7.7.7 (DNA polymerase)
17,007 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

An activity gel analysis was performed in order to examine the catalytically active component of human immunodeficiency virus (HIV) reverse transcriptase in purified enzyme preparations and HIV-infected cell extracts. Immunoaffinity purified HIV reverse transcriptase contains two proteins with molecular weights 66,000 and 51,000 in approximately equal proportions. After denaturing polyacrylamide gel electrophoresis and removal of sodium dodecyl sulfate, the p66 component of reverse transcriptase was sufficient for both DNA- and RNA-directed DNA synthesis. No DNA synthetic activity of p51 was observed. Recovery of p66 catalytic activity was approximately 10% that of DNA polymerase beta, and the density of the autoradiographic band corresponding to p66 was linear with enzyme concentration. No additional HIV-specific DNA polymerases besides p66 were observed in HIV-infected H9 cell extracts.
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PMID:Enzyme activity gel analysis of human immunodeficiency virus reverse transcriptase. 245 63

A study of steady-state kinetics of polymerization by purified human immunodeficiency virus DNA polymerase (reverse transcriptase) has been conducted. DNA synthesis was examined using a system of poly(rA) as template, oligo(dT) as primer, and dTTP as nucleotide substrate. The substrate initial velocity patterns point to an ordered mechanism with template-primer adding first. Product inhibition kinetics with either pyrophosphate or phosphonoformate are consistent with this mechanism. The human immunodeficiency virus reverse transcriptase acts processively in this replication system, but exhibits some probability of terminating after each dTMP addition to the nascent chain. The probability of terminating was approximately 20-fold higher after the first dTMP addition than after subsequent additions. With this information on the mode of polymerization, appropriate kinetic models and steady-state rate equations are discussed. In further studies, we found that a heterologous polynucleotide, poly(rC), is a potent inhibitor of the enzyme. The pattern of this inhibition is uncompetitive against template-primer, suggesting that interaction with free enzyme is not the mechanism of the inhibition.
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PMID:Studies on the mechanism of human immunodeficiency virus reverse transcriptase. Steady-state kinetics, processivity, and polynucleotide inhibition. 245 25

The development of potent anti-retroviral drugs is central to the control of human immunodeficiency virus (HIV) infection and the prevention of disease. Despite the benefit (albeit limited) shown by the early trials of zidovudine in patients with acquired immune deficiency syndrome (AIDS), there is general agreement that the best prospects for therapeutic intervention lie in the use of agents early in the infectious process. There is a definite possibility that this can be achieved if compounds acting specifically against virus encoded events can be found or developed. Although relatively simple in its structure, HIV is highly sophisticated in its mode of replication. The unique nature of the replication cycle of the retroviridae and the specific controlling mechanisms operative in HIV offer a number of possible targets for chemotherapeutic agents. The details of the structure and replication cycle of HIV will be briefly reviewed with comments on the possible virus specific and non-specific sites for potential antiviral drug development. The first specific target to be recognised was the unique, virus-associated enzyme, the reverse transcriptase (RNA directed DNA polymerase). Several inhibitors of reverse transcriptase were identified during the 1970s (e.g. suramin, HPA23, phosphonoformate). These have been found, in early trials, to be either insufficiently potent or too toxic to consider for development as anti-retroviral drugs. Indeed, knowledge of the pathogenesis of HIV infection led to the realisation that any putative drug would need to satisfy several important criteria; namely potency, low toxicity, easy administration, penetration of the blood-brain barrier and hopefully, low production costs.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Targets for antiviral therapy of human immunodeficiency virus infection. 246 49

Reverse transcriptase from the human immunodeficiency virus type I (HIV-1) was expressed in E. coli and purified to near homogeneity. The enzyme was shown to contain reverse transcriptase, DNA polymerase and ribonuclease H activities. The DNA polymerase activity converted singly-primed phi X174 (+) DNA into the double-stranded form. Two third of the replication product is ligatable to covalently closed circular DNA (RFIV-form DNA) indicating that DNA synthesis by HIV reverse transcriptase can proceed until the enzyme matches the 5'-end of a pre-existing primer molecule. The in vitro accuracy of HIV reverse transcriptase was measured with the phi X174am16 reversion assay to be 1/7,400. Reversion rates for the individual mispairs were determined from pool bias studies to be 1/8,000 for the dGMP:T template mismatch, 1/35,000 for the dGMP:A template mismatch, 1/45,000 for the dAMP:G template mismatch, 1/73,000 for the dCMP:T template mispair, 1/140,000 for the dCMP:A template mispair, and 1/180,000 for the dGMP:G template mismatch. The dTMP:T template mispair was below the detection limit of the assay indicating a reversion rate of less than 1/300,000 for this particular mispair.
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PMID:Fidelity of human immunodeficiency virus type I reverse transcriptase in copying natural DNA. 246 38

A plasmid construct expressing the p66 version of the human immunodeficiency virus reverse transcriptase as a bacterial fusion protein was subjected to in vitro mutagenesis, and the resulting variant proteins were assayed to define the locations of the two major enzymatic activities. The DNA polymerase activity was localized to the N-terminal portion of the protein; mutations altering or eliminating the C-terminal portion had little or no effect on that activity. The results suggest that, in contrast with previous reports, the p51 subunit found in virions should exhibit DNA polymerase activity. Mutations in many parts of the protein eliminated RNase H activity, suggesting that several areas are needed for proper folding and generation of that activity.
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PMID:Linker insertion mutagenesis of the human immunodeficiency virus reverse transcriptase expressed in bacteria: definition of the minimal polymerase domain. 247 90

Carbocylic 2',3'-didehydro-2',3'-dideoxyguanosine (Carbovir; NSC 614846) is an antiretroviral agent which may be useful in the treatment of AIDS. We have synthesized the 5'-triphosphate of Carbovir and examined its ability to inhibit human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (EC 2.7.7.49) and other retroviral reverse transcriptases, as well as human DNA polymerases alpha, beta, gamma (EC 2.7.7.7) and DNA primase (EC 2.7.7.6). Carbovir triphosphate emerges as a highly selective inhibitor of reverse transcriptases with little, if any, effect on the cellular enzymes. 3'-Azido-2',3'-dideoxythymidine (AZT) triphosphate and the two dideoxynucleoside triphosphates, ddTTP and ddGTP, inhibited HIV-1 reverse transcriptase to the same degree as Carbovir triphosphate, but were less selective in that they also inhibited DNA polymerases beta and gamma. We conclude that Carbovir is a highly selective antiretroviral agent.
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PMID:Comparison of the effect of Carbovir, AZT, and dideoxynucleoside triphosphates on the activity of human immunodeficiency virus reverse transcriptase and selected human polymerases. 247 36

There is considerable interest in the potential of human immunodeficiency virus type 1 (HIV-1) to develop drug resistance, especially as 3'-azido-3'-deoxythymidine (Retrovir) is now in widespread clinical use to treat people with AIDS and AIDS-related complex (ARC). To address this possibility, mutations in the HIV reverse transcriptase [deoxynucleoside-triphosphate:DNA deoxynucleotidyltransferase (RNA-directed), EC 2.7.7.49] gene have been introduced by site-directed mutagenesis of cloned constructs in Escherichia coli. Analysis of the recombinant mutant reverse transcriptase from a number of these constructs revealed enzymes that maintained enzyme activity but had a reduced ability to recognize inhibitors such as azidothymidine triphosphate. To assess the infectivity of these mutants, several constructs of proviral HIV clones with mutant reverse transcriptase genes have been made and used to transfect T cells. All five mutants tested have lower infectious potential, suggesting considerable levels of reverse transcriptase activity are required for efficient virus replication. Viable virus recovered from two clones showed decreased sensitivity to the antiviral compound phosphonoformate, thus demonstrating the potential for drug-resistant HIV to replicate. However, although the reverse transcriptase from these mutant viruses showed decreased sensitivity to azidothymidine triphosphate, paradoxically these viruses were hypersensitive to azidothymidine when tested in culture.
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PMID:Infectious potential of human immunodeficiency virus type 1 reverse transcriptase mutants with altered inhibitor sensitivity. 247 34

Human immunodeficiency virus (HIV) reverse transcriptase has been purified from yeast transformed by an autoreplicating plasmid containing the retroviral DNA polymerase gene. The previously described purification procedure for the yeast-expressed reverse transcriptase [Barr, P.J., Power, M.D., Chun Ting Lee-Ng, Gibson, H. & Luciw, P. (1987) Bio/Technology 5, 486-489] has been substantially modified, leading to an increased yield and a higher degree of purity. Several biochemical properties of the enzyme are described (template specificity, effect of DNA synthesis inhibitors); interestingly, HIV reverse transcriptase is highly resistant to N-ethylmaleimide. A complex between the human retroviral enzyme and the bovine tRNALys was shown, using a direct approach, by glycerol gradient centrifugation, as well as by the protective and specific effect of the tRNALys against enzyme inactivation by thermal denaturation and trypsin digestion. A competitive type of inhibition of HIV reverse transcriptase by tRNALys, but not by tRNAVal, is observed when viral RNA or activated DNA are used as templates.
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PMID:Human immunodeficiency virus reverse transcriptase expressed in transformed yeast cells. Biochemical properties and interactions with bovine tRNALys. 247 48

The effect of 2',3'-dideoxycytidine, a potent antiviral agent, which, following anabolic phosphorylation, inhibits the reverse transcriptase of the human immunodeficiency virus in vitro, was assessed in 16 Pekin ducks chronically infected with the duck hepatitis B virus. Nine ducks were given 11 mg/m2 of dideoxycytidine intravenously every 6 h, and 7 ducks received no treatment. Serum duck hepatitis B virus deoxyribonucleic acid and deoxyribonucleic acid polymerase activity decreased in every duck treated with dideoxycytidine. The mean inhibition of deoxyribonucleic acid polymerase and duck hepatitis B virus deoxyribonucleic acid on the third day of treatment measured 64% (p less than 0.01) and 73% (p less than 0.01), respectively. The inhibition of deoxyribonucleic acid polymerase persisted after treatment was stopped, and 4 ducks continued to show greater than 50% inhibition 12 days after stopping treatment. Duck hepatitis B virus deoxyribonucleic acid, which was measured in total cellular deoxyribonucleic acid extracted from liver biopsy specimens obtained before and on the last day of treatment with dideoxycytidine, showed an average inhibition of 96% in 3 ducks treated with dideoxycytidine, but showed no decrease in the remaining 5 ducks. Thus, dideoxycytidine has potent antiviral activity against duck hepatitis B virus and warrants further evaluation as an antiviral agent in the treatment of chronic hepatitis B virus infection in humans.
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PMID:Inhibition of duck hepatitis B virus replication by 2',3'-dideoxycytidine. A potent inhibitor of reverse transcriptase. 247 99

The unusually high error rate of human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) suggests that polymerization errors by this enzyme contribute to the genetic variability of the AIDS virus. We have analyzed the mechanism for HIV-1 RT infidelity by studying two distinct steps that might lead to base substitution mutations: nucleotide misinsertions and elongation from 3'-terminal DNA mispairs. Our results indicate that the capacity of HIV-1 RT to polymerize nucleotides onto mispaired termini is a major factor in the production of mutations by this enzyme. When a noncomplementary dAMP was inserted opposite a template adenine by HIV-1 RT, the nascent 3'-terminal A.A mispair was readily extended by subsequent incorporation of the next complementary nucleotide. The frequencies of nucleotide addition onto 3'-terminal A-A, A-C, and A-G mispairs were determined by quantitating the amount of extended primers with a gel electrophoresis assay and by measuring mutagenesis after hybridization of mismatched primers opposite an amber mutation in bacteriophage phi X174 DNA. The mispair extension frequencies are approximately 50-fold higher by HIV-1 RT than by the mammalian replicative enzyme DNA polymerase alpha.
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PMID:Extension of mismatched 3' termini of DNA is a major determinant of the infidelity of human immunodeficiency virus type 1 reverse transcriptase. 247 23


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