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

---

Query: EC:2.7.7.49 (reverse transcriptase)
31,746 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The carcinogen ethylene dibromide (EDB) has been shown to cause glutathione (GSH)-dependent base-substitution mutations, especially GC to AT transitions, in a variety of bacterial and eukaryotic systems. The known DNA adducts S-[2-(N7-guanyl)ethyl]GSH, S-[2-(N2-guanyl)ethyl]GSH, and S-[2-(O6-guanyl)ethyl]GSH were individually placed at a site in a single oligonucleotide. Polymerase extension studies were carried out using Escherichia coli polymerase I exo- (Klenow fragment, Kf-) and polymerase II exo- (pol II-), bacteriophage T7 polymerase exo-, and human immunodeficiency virus-1 reverse transcriptase in order to characterize misincorporation events. Even though extension was not as efficient as with the nonadducted template, some fully extended primers were observed with the template containing S-[2-(N7-guanyl)ethyl]GSH using all of these polymerases. dCTP was the most preferred nucleotide incorporated opposite S-[2-(N7-guanyl)ethyl]GSH by most of polymerases examined; however, dTTP incorporation was observed opposite S-[2-(N7-guanyl)ethyl]GSH with pol II-. Both S-[2-(N2-guanyl)ethyl]GSH and S-[2-(O6-guanyl)ethyl]GSH strongly blocked replication by all polymerases. Only dATP and dGTP were incorporated opposite S-[2-(N2-guanyl)ethyl]GSH by both Kf- and pol II-. S-[2-(O6-Guanyl)ethyl]GSH was shown to strongly code for dATP incorporation by Kf-. With pol II-, dTTP was incorporated opposite S-[2-(O6-guanyl)ethyl]GSH. In conclusion, all three GSH-guanyl adducts derived from the carcinogen EDB blocked the polymerases and were capable of miscoding.
...
PMID:Polymerase blockage and misincorporation of dNTPs opposite the ethylene dibromide-derived DNA adducts S-[2-(N7-guanyl)ethyl]glutathione, S-[2-(N2-guanyl)ethyl]glutathione, and S-[2-(O6-guanyl)ethyl]glutathione. 954 1

Oligodeoxynucleotides modified site-specifically with cis-thymine glycol or urea residue, two ionizing radiation/oxidation damages, were used as templates in primer extension reactions catalyzed by 3' --> 5' exonuclease-deficient Klenow fragment, human DNA polymerase beta, AMV reverse transcriptase, and a modified T7 DNA polymerase (Sequenase). Both lesions blocked DNA replication one nucleotide before and opposite the lesion site, but a significant fraction of full-length product was obtained after prolonged incubation. Hill plot analysis of the results on both thymine glycol- and urea- containing templates by 3' --> 5' exonuclease-deficient Klenow fragment for incorporation of either dATP or dGTP gave linear plots with Hill coefficients much less than 1. This suggests that the dNTP concentration influences the termination of DNA synthesis at multiple steps of the catalytic process. The specificity of nucleotide incorporation opposite these lesions and chain extension by the same polymerase was determined by a steady-state kinetic analysis. The kinetic studies established that the rate of nucleotide incorporation and chain extension was highest with deoxyadenosine opposite both these lesions. However, the efficiency of forming a G.T pair relative to an A.T pair for the control at a level of 1/10(9) was enhanced to approximately 1/160 for thymine glycol and 1/20 for urea, although the former lesion was more bypassable than the latter lesion. On the basis of these in vitro results, we conclude that both these DNA damages are impediments of DNA synthesis and that a urea residue, in particular, has the potential to miscode.
...
PMID:Replication inhibition and miscoding properties of DNA templates containing a site-specific cis-thymine glycol or urea residue. 962 35

The synthesis of enantiomerically pure carbocyclic adenosine derivatives which have been prepared based on the kinetic resolution of a trans-2-(hydroxymethyl)cyclopentanol derivative is described. Their corresponding triphosphates were evaluated as inhibitors of DNA polymerase beta, terminal deoxynucleotidyl transferase (TdT), telomerase, Escherichia coli DNA polymerase I and reverse transcriptase of human immunodeficiency virus. Surprisingly, the triphosphate of (1S,2R)-1-(6-aminopurin-9-yl)-2-(hydroxymethyl)cyclopentane [(1S,2R)-6] and its enantiomer (1R,2S)-6 emerged as strong inhibitors of TdT (Ki = 0.5 and 1.9 mM, Kmapp dATP = 40 mM), whereas the activities of all other enzymes tested proved to be unaffected.
...
PMID:Chemo-enzymatic synthesis of a new type of enantiomerically pure carbocyclic nucleoside analogues with strong inhibitory effects on terminal deoxynucleotidyl transferase. 968 Nov 36

(R)-9-(2-Phosphonylmethoxypropyl)adenine (PMPA) is an acyclic nucleoside phosphonate that has been shown to be effective in the treatment of AIDS although it has a shorter separation between the adenine and phosphorus than dideoxy-AMP and dAMP. By using pre-steady state kinetic methods, we examined the incorporation of the diphosphate of PMPA, 2',3'-dideoxyadenosine 5'-triphosphate (ddATP), and dATP catalyzed by wild-type human immunodeficiency virus type 1 (HIV-1) reverse transcriptase, an exonuclease-deficient T7 DNA polymerase (T7 exo-), and wild-type rat DNA polymerase beta in order to evaluate the selectivity of PMPA as an antiviral inhibitor. With a DNA/DNA or DNA/RNA 22/43-mer duplex, the diphosphate of PMPA (PMPApp) is as effective as ddATP in reactions catalyzed by HIV-1 reverse transcriptase in that both analogs have similar substrate specificity constants (kp/Kd) which are only 5-fold lower than dATP. In contrast, PMPApp is a much weaker inhibitor of the reaction catalyzed by T7 exo- (with the DNA/DNA 22/43-mer duplex) in that PMPApp has a 5 x 10(-4)-fold lower kp/Kd than ddATP and dATP. The lower kp/Kd of PMPApp is due to a 1000-2000-fold lower incorporation rate (kp) and a 35-45-fold lower binding constant (Kd). Similarly, PMPApp is 800-fold less inhibitory toward polymerase beta with the DNA/DNA 22/43-mer duplex, whereas in studies with a single nucleotide gapped DNA (22-20/43-mer) PMPApp is 13-fold less inhibitory than ddATP. Although parallel studies will need to be performed using appropriate human polymerases, these results begin to define the mechanistic basis for the reported lower toxicity of PMPA in the treatment of AIDS.
...
PMID:Selective inhibition of HIV-1 reverse transcriptase by an antiviral inhibitor, (R)-9-(2-Phosphonylmethoxypropyl)adenine. 976 48

2-Chloro-2'-deoxyadenine (2CldA) is used for treatment of several lymphoid malignancies. Since this drug is incorporated into DNA, we have undertaken studies on base pairing of 2-chloroadenine (2ClA). 2CldA phosphoramidite was synthesized and used for preparation of 25-mer templates with 2ClA located at site 21 from the 3'-end. Kinetic parameters (Km and Vmax) for the incorporation of deoxynucleoside-5'-triphosphates by AMV reverse transcriptase opposite the 2ClA template, as well as for the extension of 2ClA.T pair, were determined. The efficiency (Vmax/Km) of incorporation of dGTP, dCTP, and dATP opposite 2ClA is at least one order of magnitude lower than opposite unmodified A. The efficiency of incorporation of dTTP opposite 2ClA is about 30-fold lower than opposite A and extension of 2ClA.T pair is 3-fold lower than of A.T pair. From the analysis of the parameters of dTTP incorporation we conclude that formation of 2ClA.T pair is thermodynamically, but not kinetically controlled. The difference in binding energy (deltadeltaG) between 2ClA.T and A.T pairs in the environment of the polymerase active site is 2 kcal/mol. Our results indicate that the presence of 2ClA in DNA slows down replication, but does not lead to base-substitution mutations.
...
PMID:Template-directed base pairing of 2-chloro-2'-deoxyadenosine catalyzed by AMV reverse transcriptase. 982 87

HIV-1 reverse transcriptase (RT) incorporates 2'-deoxyisoguanosine triphosphate (d-isoGTP) opposite thymidine (T) in a DNA template and opposite uracil (U) in an RNA template about 10 times more efficiently than the eukaryotic DNA polymerase alpha, both in the absence and presence of dATP.
...
PMID:Recognition of 2'-deoxyisoguanosine triphosphate by HIV-1 reverse transcriptase and mammalian cellular DNA polymerases. 987 6

The majority of pre-steady-state kinetic investigations with HIV-1 reverse transcriptase (HIV-1 RT) have reported substoichiometric bursts (30-50%) of product formation in the initial reaction cycle. By using quantitative amino acid analysis, we have revised the extinction coefficient of the HIV-1 RT heterodimer and show that normal nucleotide incorporation (canonical four bases) proceeds with quantitative bursts in the first cycle. We have also modeled our previous results with this polymerase, including four situations with 8-oxo-7,8-dihydroguanine (8-oxoGua) moieties in which substoichiometric bursts (2-35%) were observed even after the correction of enzyme concentration by amino acid analysis. These include insertion of dATP opposite template 8-oxoGua, insertion of (deoxy) 8-oxoGua 5'-triphosphate opposite template C, and extension of primers beyond 8-oxoGua-A and 8-oxoGua-C pairs. The "minimal" polymerase mechanism and three others were evaluated using KINSIM and FITSIM methods. The latter three mechanisms involve a conformationally distinct, inactive polymerase-DNA-dNTP complex in equilibrium with the initial ternary complex and a conformationally distinct complex leading to phosphodiester bond formation. All three of the modified mechanisms fit the observed reaction results, but the minimal mechanism did not. Nonfunctional binary complexes (enzyme-DNA) are an alternate explanation (to ternary complexes) in some cases. Finally, DNA trapping experiments indicate that enzyme does not dissociate from the 8-oxoGua-containing DNA substrate prior to phosphodiester bond formation. We conclude that HIV-1 RT is fully active in normal nucleotide incorporation and that substoichiometric bursts with modified systems are well-described by the existence of nonproductive ternary complexes, which can isomerize to productive complexes.
...
PMID:Explanation of pre-steady-state kinetics and decreased burst amplitude of HIV-1 reverse transcriptase at sites of modified DNA bases with an additional, nonproductive enzyme-DNA-nucleotide complex. 1020 Jan 70

Hydroxyurea inhibits cellular ribonucleotide reductase, resulting in decreased pools of dNTPs and thus inhibition of DNA synthesis. Studies in vitro have shown that hydroxyurea reduces dNTP pools in cells infected with human immunodeficiency virus type 1 (HIV-1), inhibiting HIV-1 DNA synthesis in infected quiescent and activated primary human lymphocytes and macrophages. Hydroxyurea also potentiates the activity of nucleoside reverse transcriptase inhibitors (NRTIs): the activated triphosphate forms of NRTIs compete with naturally occurring dNTPs for incorporation into nascent viral DNA during reverse transcription. A synergistic effect is observed between hydroxyurea and didanosine (2',3'-dideoxyinosine; DDI). This combination exerts persistent suppression of HIV-1 replication without evidence of viral rebound for over 1 year in HIV-1-infected patients. Didanosine-resistant HIV-1 mutants retain sensitivity to didanosine in the presence of hydroxyurea. The incorporation of didanosine triphosphate by resistant reverse transcriptase is increased in the context of the hydroxyurea-induced depletion of dATP. Although hydroxyurea has a reduced effect on dNTPs competing with the triphosphate forms of pyrimidine NRTIs, it appears to augment the anti-HIV-1 activity of these agents by increasing their intracellular phosphorylation; this may be of particular interest for salvage strategies given recent data indicating disruption of NRTI phosphorylation with specific NRTI treatment regimens. Finally, by exerting a cytostatic effect on CD4 and CD8 T lymphocytes, hydroxyurea may (i) reduce HIV-1 replication by decreasing CD4 T cell proliferation; and (ii) prevent the exhaustion of CD8 T cell populations that may occur as a result of excessive activation in the context of HIV-1 infection.
...
PMID:Hydroxyurea: mechanisms of HIV-1 inhibition. 1072 6

Certain drugs used in the treatment of lung cancer and other human malignancies are cytotoxic because of their ability to interact with the two isoforms of topoisomerase II (topo II), topo IIalpha and topo IIbeta. As part of an effort to evaluate the contribution of topo II alterations to drug sensitivity and resistance in lung cancer, we have developed a semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) assay to measure levels of topo II alpha and beta mRNAs simultaneously using a single pair of primers with sequences common to both isoforms. The PCR products derived from the topo II alpha and beta mRNAs are both 446 bp but have different electrophoretic mobilities in a nondenaturing polyacrylamide gel, allowing sensitive, rapid quantitation when the products are radiolabeled with [35S]-dATP. Using this RT-PCR method, poly(A+) RNA from 13 non-small cell lung cancer (NSCLC) cell lines was analyzed. The results obtained indicated that the cell lines express a wide range of topo II alpha mRNA levels (12-fold) and topo IIbeta mRNA levels (5.5-fold). Tumor and normal lung tissues from 25 patients with NSCLC were also examined. In the tumor samples, the levels of the topo II alpha and beta mRNAs were similar. However, mean topo IIalpha mRNA levels in the tumors were approximately 7-fold higher than those of the paired normal lung tissues. In contrast, topo IIbeta mRNA levels were similar in both tumor and normal lung. Topo II alpha and beta mRNA levels were both significantly lower in the squamous cell tumors than in the adenocarcinoma samples. Topo IIbeta mRNA levels in the squamous cell tumors were also significantly lower than those in paired normal lung tissue. The RT-PCR method described is reliable and convenient, and for the first time, makes the rapid simultaneous direct comparison of topo IIalpha and topo IIbeta mRNA levels feasible in large numbers of clinical samples.
...
PMID:Simultaneous quantitation of topoisomerase II alpha and beta isoform mRNAs in lung tumor cells and normal and malignant lung tissue. 1087 30

The fidelity of the yeast retrotransposon Ty1 reverse transcriptase (RT) was determined by an assay based on gel electrophoresis. Steady-state kinetics analyses of deoxyribonucleotide (dNTP) incorporation at a defined primer-template site indicate that Ty1 RT misincorporates dNTP at a frequency of 0.45 x 10(-5) for the A(t):A mispair in which dATP is misincorporated opposite a template A to 6.27 x 10(-5) for the C(t):A mispair. The G(t):G and T(t):T mispairs are formed with very low efficiency. The fidelity parameters of Ty1 RT do not depend on whether RNA or DNA are copied. Relative to lentiviral RTs (HIV-1, HIV-2 or EIAV) Ty1 RT is approximately 10-fold less error prone. Our data also show that the Ty1 RT is able to recapitulate two error-generating mechanisms: extension of mismatches and non-templated addition of nucleotides at the end of a blunt-end primer-template.
...
PMID:DNA synthesis fidelity by the reverse transcriptase of the yeast retrotransposon Ty1. 1137 39


<< Previous 1 2 3 4 5 6 7 8 Next >>

---