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)

4'-Azidothymidine (ADRT) is a novel nucleoside analogue that exhibits potent inhibitory activity against the replication of human immunodeficiency virus (HIV) in lymphocytes. The mechanisms by which ADRT inhibits HIV reverse transcriptase (HIV-RT) as ADRT 5'-triphosphate (ADRT-TP), the active intracellular metabolite of ADRT, and as the ADRT-MP molecule incorporated into DNA were examined and compared to their effects on human DNA polymerases alpha and beta. Inhibition of HIV-RT by ADRT-TP is competitive against TTP and is more potent against RNA to DNA synthesis (Ki = 0.009 microM versus Km = 3.3 microM for TTP) than it is against DNA to DNA synthesis (Ki = 0.95 microM versus Km = 16.3 microM for TTP). ADRT-TP is also a more potent inhibitor for primer elongation on RNA template than on DNA template. ADRT-TP is a poor inhibitor of human DNA polymerases alpha (Ki = 62.5 microM) and beta (Ki = 150 microM) (Chen et al., 1992). The consequences of ADRT incorporation into DNA are strikingly different for the HIV-RT and for human DNA polymerases alpha and beta. DNA polymerases alpha and beta incorporate a single ADRT-MP molecule into nascent DNA at a very slow rate and continue to elongate. They are unable to incorporate a second consecutive ADRT-MP. However, HIV-RT is able to efficiently incorporate two consecutive ADRT molecules. Incorporation of two consecutive ADRT-MP molecules by HIV-RT prevents further DNA chain elongation. Incorporation of two ADRT-MP molecules separated by one deoxyribonucleoside monophosphate (dAMP, dCMP, or dGMP) also abolishes DNA chain elongation by HIV-RT.
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PMID:Selective action of 4'-azidothymidine triphosphate on reverse transcriptase of human immunodeficiency virus type 1 and human DNA polymerases alpha and beta. 768 86

The alpha-anomer of deoxyadenosine (alpha-dA) is a major adenine lesion produced by hydroxyl radicals in DNA. To assess its biochemical effects on DNA replication, alpha-dA was site-specifically incorporated into oligodeoxyribonucleotide templates using phosphoramidite chemistry. alpha-dA in the template constituted a transient block to DNA synthesis catalyzed by Escherichia coli DNA polymerase I Klenow fragment (polI), but translesional synthesis occurred after prolonged incubation. Primer extension assays and Maxam-Gilbert sequencing of newly synthesized products revealed that alpha-dA directed not only incorporation of the correct nucleotide, dTMP, opposite the lesion but also misincorporation of dAMP and dCMP. dGMP was barely incorporated under these conditions. The order of the incorporation frequency at the alpha-dA site was affected by the nearest neighbor base pair 3' to the lesion. T7 and Taq DNA polymerases, as well as RAV-2 reverse transcriptase, showed a selectivity similar to that of PolI with respect to the nucleotide incorporation opposite alpha-dA, suggesting that the discrimination of nucleotides associated with alpha-dA is independent of the origin of DNA polymerases and is an intrinsic feature of the lesion. The mutational spectrum predicted for alpha-dA (i.e., A-->G transitions and A-->T transversions) is significantly different from those reported for other hydroxyl radical induced DNA lesions such as abasic sites or 7,8-dihydro-8-oxoguanine, both primarily directing misincorporation of A. Possible biological consequences and the mechanism of dNTP discrimination associated with alpha-dA are discussed.
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PMID:Replication of DNA templates containing the alpha-anomer of deoxyadenosine, a major adenine lesion produced by hydroxyl radicals. 800 79

Rauscher murine leukemia virus induces an erythroleukemia in susceptible strains of mice that is associated with splenomegaly and viremia. This animal model has been used for evaluating the in vivo efficacy of potential anti-HIV agents. The in vivo antiviral activity of therapeutic agents has usually been determined by measuring a reduction in the spleen weights of compound-treated mice or by quantitating viremia with the UV-XC plaque assay. The UV-XC assay, however, is time-consuming and labor-intensive. Virions of Rauscher murine leukemia virus, like other retroviruses, contain the enzyme reverse transcriptase. Quantitating the level of this enzyme in infected mouse sera provides a more rapid measure of viremia in the animal. We have examined the effects of several reagents, including detergent, KCl, EGTA, dGMP, spermine, as well as protease and RNase inhibitors, on the reverse transcriptase assay. The optimized assay method was effective in evaluating the antiviral activity of AZT in the Rauscher murine leukemia virus in vivo model. The assay is also amenable to automation if large numbers of assays are required.
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PMID:Optimization of the reverse transcriptase assay for the detection of viral burden in mice infected with Rauscher murine leukemia virus. 891 Jun 49

We have used 8-O-dGTP, a mutagenic nucleotide generated by oxidative metabolism, to probe the misincorporation potential of HIV-1 reverse transcriptase (RT) during DNA synthesis templated by the same nucleotide sequence as either RNA or DNA. With either template, 8-O-dGMP was misincorporated opposite template A, yielding characteristic A-->C transversions. The error rate with DNA was similar to that with RNA, suggesting that base misincorporation by the RT during first-strand and second-strand replication may contribute equally to the HIV-1 base substitution mutation rate. The rate of 8-O-dGMP misincorporation differed by more than 10-fold among the 20 adenines in the M13mp2 template where A-->C transversions can be detected. The transversion distribution was similar with the two templates, indicating that the effects of flanking nucleotides on misincorporation rates were similar. This is consistent with structural and biochemical data suggesting that HIV-1 RT binds RNA x DNA and DNA x DNA template-primers in the same orientation. The similarities in error rates and distribution further indicate that, despite differences in the structures of free RNA x DNA and DNA x DNA duplexes (e.g., minor groove dimensions), the polymerase active site that assembles upon substrate binding establishes a similar degree of nucleotide selectivity with both types of template-primers. Comparison of the RT error distribution to that observed with two Pol I family DNA polymerases and a Pol alpha family polymerase revealed common hot and cold spots for misincorporation. This suggests that the local nucleotide sequence influences the nucleotide selectivity of four polymerases in a similar manner, despite their differences in structure, biochemical properties, and functions.
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PMID:The base substitution fidelity of HIV-1 reverse transcriptase on DNA and RNA templates probed with 8-oxo-deoxyguanosine triphosphate. 1052

Telomerase is a reverse transcriptase that adds single-stranded telomeric repeats to the ends of linear eukaryotic chromosomes. It consists of an RNA molecule including a template sequence, a protein subunit containing reverse transcriptase motifs, and auxiliary proteins. We have carried out an interference footprinting analysis of the Tetrahymena telomerase elongation complexes. In this study, single-stranded oligonucleotide primers containing telomeric sequences were modified with base-specific chemical reagents and extended with the telomerase by a single (32)P-labeled dGMP or dTMP. Base modifications that interfered with the primer extension reactions were mapped by footprinting. Major functional interactions were detected between the telomerase and the six or seven 3'-terminal residues of the primers. These interactions occurred not only with the RNA template region, but also with another region in the enzyme ribonucleoprotein complex designated the telomerase DNA interacting surface (TDIS). This was indicated by footprints generated with dimethyl sulfate (that did not affect Watson-Crick hydrogen bonding) and by footprinting assays performed with mutant primers. In primers aligned at a distance of 2 nucleotides along the RNA template region, the footprints of the six or seven 3'-terminal residues were shifted by 2 nucleotides. This shift indicated that during the elongation reaction, TDIS moved in concert with the 3' ends of the primers relative to the template region. Weak interactions occurred between the telomerase and residues located upstream of the seventh nucleotide. These interactions were stronger in primers that were impaired in the ability to align with the template.
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PMID:Interference footprinting analysis of telomerase elongation complexes. 1082 87

A site-specifically modified oligonucleotide containing a single 2'-deoxyribonolactone lesion was used as a template for primer extension reactions catalyzed by M-MuLV reverse transcriptase (RT) and by the Klenow fragments of Escherichia coli DNA polymerase proficient (KF exo(+)) or deficient (KF exo(-)) in exonuclease activity. Analysis of the extension products in the presence of the four dNTPs or of a single dNTP showed that the M-MuLV RT was completely blocked and did not incorporate any dNMP opposite 2'-deoxyribonolactone. KF exo(-) preferentially incorporated nucleotides opposite the lesion following the frequency order dAMP > dGMP >> dTMP approximately dCMP and thus appeared to obey the 'A rule' for preferential incorporation as has been shown previously for the 2'-deoxyribose abasic site. In the sequence context examined, the primer extension by KF exo(-) appeared to be less efficient when dAMP was positioned opposite the lesion as compared with dTMP or dGMP. These two nucleotides promoted a more efficient polymerization accompanied by nucleotide deletion through misalignment incorporations. We therefore predict that the sequence context may strongly influence the translesional synthesis by KF exo(-) and thus the miscoding and mutational potential of the 2'-deoxyribonolactone in E.coli.
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PMID:Translesional synthesis on DNA templates containing the 2'-deoxyribonolactone lesion. 1143 17

The reverse transcriptase of the human immunodeficiency virus type 1 (HIV-1 RT) does not possess an exonucleolytic proofreading activity; however, previous studies have shown that this enzyme can excise incorporated chain-terminators in the presence of pyrophosphate or ATP. This type of reaction provides a plausible mechanism for HIV-1 resistance to several nucleoside analogue inhibitors. Here we studied the efficiency of pyrophosphorolysis in the context of mismatched nucleotides, and found that the removal of dCMP and dTMP opposite T is literally blocked. Thus, pyrophosphorolysis may not provide an alternative, universal proofreading mechanism, although excision of dGMP and the correct dAMP opposite T can occur with considerable efficiency. Site-specific footprinting experiments revealed that the 3' end of C:T- and T:T-mispaired primer strands is predominantly found in a post-translocational configuration, which prevents the removal of terminal nucleotides. In contrast, complexes containing G:T and A:T base pairs can exist in both post- and pre-translocational stages. Excision can only occur in the latter, which helps to explain the observed selectivity of the reaction. The efficiency of mismatch extensions does not appear to depend on pre-existing changes of the translocational equilibrium. However, footprints of complexes containing 3' penultimate mismatches suggest that the incorporation of the first nucleotide following the mispair can force the enzyme to slide backwards, which can inhibit ensuing polymerization events. The fact that misincorporated nucleotides can affect the precise positioning of RT provides a rational for the development of novel nucleoside analogue inhibitors that contain modifications in the base moiety.
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PMID:Impact of the translocational equilibrium of HIV-1 reverse transcriptase on the efficiency of mismatch extensions and the excision of mispaired nucleotides. 1518 47

We recently identified, from the gamma-irradiation mixture of duplex DNA, a new intrastrand G[8-5]C cross-link lesion, in which the C8 atom of guanine and the C5 atom of its 3' neighboring cytosine are covalently bonded, and carried out in vitro replication studies for the lesion-bearing substrate with a translesion synthesis polymerase, yeast polymerase eta. Here we extended the in vitro replication studies to two replicative polymerases, exonuclease-deficient bacteriophage T7 DNA polymerase (T7(-)) and HIV reverse transcriptase (HIV-RT). Primer extension assays showed that both polymerases stopped synthesis after incorporating a nucleotide opposite the 3'-cytosine in the G[8-5]C lesion. Steady-state kinetic measurements for nucleotide incorporation opposite the 3'-cytosine of the lesion showed that both T7(-) and HIV-RT preferentially incorporated the correct nucleotide, dGMP. We also examined the thermal stabilities and base pairing properties of G[8-5]C in d(ATGGCG[8-5]CGCTAT). The G[8-5]C lesion destabilizes the duplex form by approximately 4 kcal/mol in free energy at 25 degrees C relative to the undamaged parent duplex, and the thermally most stable duplex has natural bases opposite the lesion.
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PMID:Thermodynamic and in vitro replication studies of an intrastrand G[8-5]C cross-link lesion. 1595 95

The synthesis of a caged RNA phosphoramidite building block containing the oxidatively damaged base 5-hydroxycytidine (5-HOrC) has been accomplished. To determine the effect of this highly mutagenic lesion on complementary base recognition and coding properties, this building block was incorporated into a 12-mer oligoribonucleotide for T(m) and CD measurements and a 31-mer template strand for primer extension experiments with HIV-, AMV- and MMLV-reverse transcriptase (RT). In UV-melting experiments, we find an unusual biphasic transition with two distinct T(m)'s when 5-HOrC is paired against a DNA or RNA complement with the base guanine in opposing position. The higher T(m) closely matches that of a C-G base pair while the lower is close to that of a C-A mismatch. In single nucleotide extension reactions, we find substantial misincorporation of dAMP and to a lesser extent dTMP, with dAMP almost equaling that of the parent dGMP in the case of HIV-RT. A working hypothesis for the biphasic melting transition does not invoke tautomeric variability of 5-HOrC but rather local structural perturbations of the base pair at low temperature induced by interactions of the 5-HO group with the phosphate backbone. The properties of this RNA damage is discussed in the context of its putative biological function.
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PMID:Synthesis, base pairing properties and trans-lesion synthesis by reverse transcriptases of oligoribonucleotides containing the oxidatively damaged base 5-hydroxycytidine. 2185 26

Multiple clinical trials have demonstrated that herpes simplex virus 2 (HSV-2) suppressive therapy using acyclovir (ACV) or valacyclovir in HIV-1/HSV-2-infected persons increased the patient's survival and decreased the HIV-1 load. It has been shown that the incorporation of ACV-monophosphate into the nascent DNA chain instead of dGMP results in the termination of viral DNA elongation and directly inhibits laboratory strains of HIV-1. We evaluated here the anti-HIV activity of ACV against primary HIV-1 isolates of different clades and coreceptor specificity and against viral isolates resistant to currently used drugs, including zidovudine, lamivudine, nevirapine, a combination of nucleoside reverse transcriptase inhibitors (NRTIs), a fusion inhibitor, and two protease inhibitors. We found that, at clinically relevant concentrations, ACV inhibits the replication of these isolates in human tissues infected ex vivo. Moreover, addition of ribavirin, an antiviral capable of depleting the pool of intracellular dGTP, potentiated the ACV-mediated HIV-1 suppression. These data warrant further clinical investigations of the benefits of using inexpensive and safe ACV alone or in combination with other drugs against HIV-1, especially to complement or delay highly active antiretroviral therapy (HAART) initiation in low-resource settings.
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PMID:Exploiting the anti-HIV-1 activity of acyclovir: suppression of primary and drug-resistant HIV isolates and potentiation of the activity by ribavirin. 2231 23


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