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)

The N-2 atom of guanine (G) is susceptible to modification by various carcinogens. Oligonucleotides with increasing bulk at this position were analyzed for fidelity and catalytic efficiency with the processive DNA polymerases human immunodeficiency virus, type 1, reverse transcriptase (RT), and bacteriophage T7 exonuclease(-) (T7(-)). RT and T7(-) effectively bypassed N(2)-methyl(Me)G and readily extended primers but were strongly blocked by N(2)-ethyl(Et)G, N(2)-isobutylG, N(2)-benzylG, and N(2)-methyl(9-anthracenyl)G. Steady-state kinetics of single nucleotide incorporation by RT and T7(-) showed a decrease of 10(3) in k(cat)/K(m) for dCTP incorporation opposite N(2)-MeG and a further large decrease opposite N(2)-EtG. Misincorporation frequency was increased 10(2)-10(3)-fold by a Me group and another approximately 10(3)-fold by an Et group. dATP was preferentially incorporated opposite bulky N(2)-alkylG molecules. N(2)-MeG attenuated the pre-steady-state kinetic bursts with RT and T7(-), and N(2)-EtG eliminated the bursts. Large elemental effects with thio-dCTP(alphaS) were observed with N(2)-EtG (6- and 72-fold decreases) but were much less with N(2)-MeG, indicating that the N(2)-Et group may affect the rate of the chemistry step (phosphodiester bond formation). Similar values of K(d(dCTP)) and K(d(DNA)) and k(off) rates of DNA substrates from RT and T7(-) indicate that ground-state binding and dissociation rates are not considerably affected by the bulk. We conclude that even a Me group at the guanine N-2 atom can cause a profound interfering effect on the fidelity and efficiency; an Et or larger group causes preferential misincorporation and strong blockage of replicative polymerases, probably at and before the chemistry step, demonstrating the role of bulk in DNA lesions.
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PMID:Analysis of the effect of bulk at N2-alkylguanine DNA adducts on catalytic efficiency and fidelity of the processive DNA polymerases bacteriophage T7 exonuclease- and HIV-1 reverse transcriptase. 1498 30

Mechanisms governing viral replicative capacity are poorly understood at the biochemical level. Human immunodeficiency virus, type 1 reverse transcriptase (HIV-1 RT) K65R or L74V substitutions confer viral resistance to 2',3'-dideoxyinosine (ddI) in vivo. The two substitutions never occur together, and L74V is frequently found in patients receiving ddI, while K65R is not. Here we show that recombinant viruses carrying K65R and K65R/L74V display the same resistance level to ddI (about 9.5-fold) relative to wild type. Consistent with this result, purified HIV-1 RT carrying K65R RT or K65R/L74V substitutions exhibits an 8-fold resistance to ddATP as judged by pre-steady state kinetics of incorporation of a single nucleotide into DNA. Resistance is due to a selective decrease of the catalytic rate constant k(pol): 22-fold (from 7.2 to 0.33 s(-1)) for K65R RT and 84-fold (from 7.2 to 0.086 s(-1)) for K65R/L74V RT. However, the K65R/L74V virus replication capacity is severely impaired relative to that of wild-type virus. This loss of viral fitness is correlated to a poor ability of K65R/L74V RT to use natural nucleotides relative to wild-type RT: 15% that of wild-type RT for dATP, 36% for dGTP, 50% for dTTP, and 25% for dCTP. The order of incorporation efficiency is wild-type RT > L74V RT > K65R RT > K65R/L74V RT. Processivity of DNA synthesis remains unaffected. These results explain why the two mutations do not combine in the clinic and might give a mechanism for a decreased viral fitness at the molecular level.
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PMID:A loss of viral replicative capacity correlates with altered DNA polymerization kinetics by the human immunodeficiency virus reverse transcriptase bearing the K65R and L74V dideoxynucleoside resistance substitutions. 1504 78

Emtricitabine [(-)FTC; (-)-beta-L-2'-3'-dideoxy-5-fluoro-3'-thiacytidine] is an oxathiolane nucleoside analog recently approved by the Food and Drug Administration for the treatment of human immunodeficiency virus (HIV). Structurally, (-)FTC closely resembles lamivudine [(-)3TC] except that the former is 5-fluorinated on the cytosine ring. In HIV-1 reverse transcriptase (RT) enzymatic assays, the triphosphate of (-)FTC [(-)FTC-TP] was incorporated into both DNA-DNA and DNA-RNA primer-templates nearly 3- and 10-fold more efficiently than (-)3TC-TP. Animal studies and clinical trial studies have demonstrated a favorable safety profile for (-)FTC. However, a detailed study of the incorporation of (-)FTC-TP by human mitochondrial DNA polymerase gamma, a host enzyme associated with nucleoside toxicity, is required for complete understanding of the molecular mechanisms of inhibition and toxicity. We studied the incorporation of (-)FTC-TP and its enantiomer (+)FTC-TP into a DNA-DNA primer-template by recombinant human mitochondrial DNA polymerase in a pre-steady-state kinetic analysis. (-)FTC-TP was incorporated 2.9 x 10(5)-, 1.1 x 10(5)-, 1.6 x 10(3)-, 7.9 x 10(3)-, and 100-fold less efficiently than dCTP, ddCTP, (+)3TC-TP, (+)FTC-TP, and (-)3TC-TP, respectively. The rate of removal of (-)FTC-MP from the corresponding chain-terminated 24-mer DNA by polymerase gamma's 3'-->5' exonuclease activity was equal to the removal of (+)FTC-MP, 2-fold slower than the removal of (-)3TC-MP and (+)3TC-MP, and 4.6-fold slower than the excision of dCMP. These results demonstrate that there are clear differences between HIV-1 RT and polymerase gamma in terms of preferences for substrate structure.
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PMID:Relationship between antiviral activity and host toxicity: comparison of the incorporation efficiencies of 2',3'-dideoxy-5-fluoro-3'-thiacytidine-triphosphate analogs by human immunodeficiency virus type 1 reverse transcriptase and human mitochondrial DNA polymerase. 1504 33

Hepadnavirus DNA polymerase functions in DNA synthesis and encapsidation, and acts as a primer for minus-strand DNA synthesis. Through protein priming reaction, a short DNA oligomer synthesized from the bulge of epsilon as template is covalently attached to the Tyr residue in the terminal protein (TP) domain of DNA polymerase. Using endogenous polymerase assays and native agarose gel analysis, we detected endogenous polymerase activity in priming-deficient mutant core particles, but not in reverse transcriptase (RT) reaction- or P protein-deficient mutant core particles. In addition, priming-deficient mutant core particles incorporated radiolabeled (32)P-dATP, (32)P-TTP, and (32)P-dGTP, but not (32)P-dCTP. Our results suggest that the priming-deficient mutant P protein has the ability to synthesize oligomers (presumably nascent minus-strand DNA) in the absence of covalent linkage between TP and the first deoxynucleotide. We propose that the priming-deficient mutant may be defective in minus-strand DNA translocation to direct repeat (DR) 1 at the 3' end of pregenomic RNA (pgRNA) that leads to the elongation of minus-strand DNA.
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PMID:Oligomer synthesis by priming deficient polymerase in hepatitis B virus core particle. 1506 13

In the absence of the viral vif gene, human immunodeficiency virus (HIV) may be restricted by the APOBEC3G gene on chromosome 22. The role of the HIV Vif protein is to exclude host cell APOBEC3G from the budding virion. As APOBEC3G shows sequence homology to cytidine deaminases, it is presumed that in the absence of Vif, cytidine residues in the cDNA are deaminated yielding uracil. It is not known if additional proteins mediate APOBEC3G function or if deamination occurs in concert with reverse transcription. This report describes an in vitro assay showing that Baculovirus derived APOBEC3G alone extensively deaminates cDNA independently of reverse transcriptase. It reproduces the dinucleotide context typical of G --> A hypermutants derived from a Delta(vif) virus. By using an RNaseH- form of reverse transcriptase, it was shown that the cDNA has to be free of its RNA template to allow deamination. APOBEC3G deamination of dC or dCTP was not detected. In short, APOBEC3G is a single-stranded DNA cytidine deaminase capable of restricting retroviral replication.
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PMID:APOBEC3G is a single-stranded DNA cytidine deaminase and functions independently of HIV reverse transcriptase. 1512 99

Six oligonucleotides with carcinogen derivatives bound at the N2 atom of deoxyguanosine were prepared, including adducts derived from butadiene, acrolein, crotonaldehyde, and styrene, and examined for effects on the replicative enzymes bacteriophage DNA polymerase T7- (T7-) and HIV-1 reverse transcriptase for comparison with previous work on smaller DNA adducts. All of these adducts strongly blocked dCTP incorporation opposite the adducts. dATP was preferentially incorporated opposite the acrolein and crotonaldehyde adducts, and dTTP incorporation was preferred at the butadiene- and styrene-derived adducts. Steady-state kinetic analysis indicated that the reduced catalytic efficiency with adducted DNA involved both an increased Km and attenuated kcat. Fluorescence estimates of Kd and pre-steady-state kinetic measurements of koff showed no significantly decreased affinity of T7- with the adducted oligonucleotides or the dNTP. Pre-steady-state kinetics showed no burst phase kinetics for dNTP incorporation with any of the modified oligonucleotides. These results indicate that phosphodiester bond formation or a conformational change of the enzyme.DNA complex is rate-limiting instead of the step involving release of the oligonucleotide. Thio elemental effects for dNTP incorporation were generally relatively small but variable, indicating that the presence of adducts may sometimes make phosphodiester bond formation rate-limiting but not always.
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PMID:Kinetics of nucleotide incorporation opposite DNA bulky guanine N2 adducts by processive bacteriophage T7 DNA polymerase (exonuclease-) and HIV-1 reverse transcriptase. 1553 46

A series of six oligonucleotides with dihydrodiol epoxide metabolites of the polycyclic aromatic hydrocarbons (PAHs) benz[a]anthracene and benzo[a]pyrene attached to adenine N6 and guanine N2 atoms were prepared and studied with the processive bacteriophage DNA polymerase T7, exonuclease- (T7-). HIV-1 reverse transcriptase was much less efficient in polymerization than T7-. Benz[a]anthracene and benzo[a]pyrene adducts strongly blocked incorporation of dTTP and dCTP opposite the A and G derivatives, respectively. dATP was preferentially incorporated in all cases. Steady state kinetic analysis indicated that the low catalytic efficiency with adducted DNA was due to both increased K(m) and lowered k(cat) values. Some differences due to PAH stereochemistry were observed. Fluorescence estimates of K(d) and presteady state kinetic measurements of k(off) showed no major decrease in the affinity of T7- with damaged DNA substrates or with dNTPs. Presteady state kinetics showed a lack of the normal burst kinetics for dNTP incorporation with all PAH-DNA derivatives. These results indicate that the rate-limiting step is at or before the step of phosphodiester bond formation; release of the oligonucleotide is no longer the slowest step. Thio elemental effects (substitution of alpha-oxygen with sulfur) were relatively small, in contrast to previous work with T7- and 8-oxo-7,8-dihydroguanine. The effect of these bulky PAH adducts is either to attenuate rates of conformational changes or to introduce an additional conformation problem but not to alter the inherent affinity of the polymerase for DNA or dNTPs.
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PMID:Kinetics of nucleotide incorporation opposite polycyclic aromatic hydrocarbon-DNA adducts by processive bacteriophage T7 DNA polymerase. 1572 Jan 47

Emtricitabine (FTC) and lamivudine (3TC) are deoxycytidine analogues with potent and selective inhibition of human immunodeficiency virus (HIV) and hepatitis B virus (HBV) replication. The K65R mutation in the HIV reverse transcriptase (RT) confers reduced susceptibility to 3TC, ddC, ddI, abacavir, and tenofovir in vitro. The Q151M mutation confers reduced susceptibility to many of the approved anti-HIV nucleoside analogues with the exception of 3TC and tenofovir. The double mutation K65R/Q151M has been shown to be more resistant to many NRTIs than either of the single mutations alone. In this study, we measured the antiviral activity of FTC and 3TC against HIV-1 containing K65R, Q151M, and K65R/Q151M mutations. We also studied the steady-state kinetic properties for the inhibition of dCTP incorporation by FTC 5'-triphosphate (TP) and 3TC-TP In addition, we measured the incorporation of dCTP, FTC-TP, and 3TC-TP into a random sequence DNA/DNA primer/template by the HIV-1 RTs using pre-steady-state kinetic analysis. Finally, we studied the incorporation of these deoxycytidine analogues into a HIV-1 genomic DNA/DNA primer/template by K65R HIV-1 RT to address certain concerns associated with DNA sequence specificity. Overall, this study demonstrated that K65R and K65R/Q151M related drug resistance to FTC and 3TC was mainly due to a significant decrease in the rate of incorporation. There was little to no effect on the binding affinities of the mutant HIV-1 RTs for the deoxycytidine analogues. The Q151M mutation remained sensitive to both FTC and 3TC in both cell culture and enzymatic assays. At a molecular level, FTC-TP was incorporated at least as efficiently as 3TC-TP for all of the HIV-1 RT and primer/templates tested.
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PMID:Virologic and enzymatic studies revealing the mechanism of K65R- and Q151M-associated HIV-1 drug resistance towards emtricitabine and lamivudine. 1644 Sep 88

To investigate how structural changes in the amino acid side chain affect nucleotide substrate selection in human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT), a variety of non-natural tyrosine analogues were substituted for Tyr115 of p66 RT. RT variants containing meta-Tyr, nor-Tyr, aminomethyl-Phe, and 1- and 2-naphthyl-Tyr were produced in an Escherichia coli coupled transcription/translation system. Mutant p66 subunits were reconstituted with wild-type (WT) p51 RT and purified by affinity chromatography. Each modified enzyme retained DNA polymerase activity following this procedure. Aminomethyl-Phe115 RT incorporated dCTP more efficiently than the WT and was resistant to the chain terminator (-)-beta-2',3'-dideoxy-3'-thiacytidine triphosphate (3TCTP) when examined in a steady-state fidelity assay. However, 2-naphthyl-Tyr115 RT inefficiently incorporated dCTP at low concentrations and was kinetically slower with all dCTP analogues tested. Models of RT containing these side chains suggest that the aminomethyl-Phe115 substitution provides new hydrogen bonds through the minor groove to the incoming dNTP and the template residue of the terminal base pair. These hydrogen bonds likely contribute to the increased efficiency of dCTP incorporation. In contrast, models of HIV-1 RT containing 2-naphthyl-Tyr115 reveal significant steric clashes with Pro157 of the p66 palm subdomain, necessitating rearrangement of the active site.
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PMID:Investigating the "steric gate" of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase by targeted insertion of unnatural amino acids. 1727 99

Recent studies have identified a role for mutations in the connection and RNase H domains of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) resistance to nucleoside analog RT inhibitors (NRTI). To provide insight into the biochemical mechanism(s) involved, we investigated the effect of the G333D mutation in the connection domain of RT on resistance to zidovudine (AZT) and lamivudine (3TC) in enzymes that contain both M184V and thymidine analog mutations (TAMs; M41L, L210W, and T215Y). Our results from steady-state kinetic, pre-steady-state kinetic, and thermodynamic analyses indicate that G333D facilitates dual resistance to AZT and 3TC in two ways. First, in combination with M184V, G333D increased the ability of HIV-1 RT to effectively discriminate between the normal substrate dCTP and 3TC-triphosphate. Second, G333D enhanced the ability of RT containing TAMs and M184V to bind template/primer terminated by AZT-monophosphate (AZT-MP), thereby restoring ATP-mediated excision of AZT-MP under steady-state assay conditions. This study is the first to elucidate a molecular mechanism whereby a mutation in the connection domain of RT can affect NRTI susceptibility at the enzyme level.
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PMID:Mechanisms by which the G333D mutation in human immunodeficiency virus type 1 Reverse transcriptase facilitates dual resistance to zidovudine and lamivudine. 1796 7


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