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 RNA genome of the lentivirus human immunodeficiency virus type 1 (HIV-1) is significantly richer in adenine nucleotides than the statistically equal distribution of the four different nucleotides that is expected. This compositional bias may be due to the guanine-to-adenine (G-->A) nucleotide hypermutation of the HIV genome, which has been explained by dCTP pool imbalances during reverse transcription. The adenine nucleotide bias together with the poor fidelity of HIV-1 reverse transcriptase markedly enhances the genetic variation of HIV and may be responsible for the rapid emergence of drug-resistant HIV-1 strains. We have now attempted to counteract the normal mutational pattern of HIV-1 in response to anti-HIV-1 drugs by altering the endogenous deoxynucleoside triphosphate pool ratios with antimetabolites in virus-infected cell cultures. We showed that administration of these antimetabolic compounds resulted in an altered drug resistance pattern due to the reversal of the predominant mutational flow of HIV (G-->A) to an adenine-to-guanine (A-->G) nucleotide pattern in the intact HIV-1-infected lymphocyte cultures. Forcing the virus to change its inherent nucleotide bias may lead to better control of viral drug resistance development.
...
PMID:Exploitation of the low fidelity of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase and the nucleotide composition bias in the HIV-1 genome to alter the drug resistance development of HIV. 1139 May 79

Substrate properties of the earlier synthesized and characterized dCTP derivatives bearing in the exo-N-position of cytosine 2-(4-azido-2,3,5,6-tetrafluorobenzoylamino)ethyl (I), 2-(2-nitro-5-azidobenzoylamino)ethyl (II), 2-(4-azido-2,3,5,6-tetrafluorobenzylideneaminooxymethylcarbonylamino)ethyl (III), 4-(4-azido-2,3,5,6-tetrafluorobenzylideneaminooxy)butyloxy (IV), or 4-(4-azido-2,3,5,6-tetrafluorobenzylidenehydrazinocarbonyl)butyl- carbonylamino (V) groups were studied in the primer extension reaction catalyzed by rat DNA polymerase beta. Unlike the earlier results obtained with HIV reverse transcriptase, dCTP derivatives (I)-(III) were not recognized by rat DNA polymerase beta as dTTP analogues, and all the five nucleotides were utilized as dCTP analogues. When compared with dCTP, Km values for the synthesized dCTP derivatives were higher by a factor of 4-20; Vmax were 1-2.3 times higher for (I)-(III) and (V) but 20-fold lower for derivative (IV). Site-specific photomodifications of the primer-template-DNA polymerase beta complexes were carried out using photoreactive reagents PRI-PRV, obtained in situ by extension of 5'-32P-labeled primers with dCTP analogues (I)-(V), respectively, when exposed to UV irradiation at 303-313 nm. Reagents PRI and PRIV provided the maximum photocrosslinking of the 5'-32P-labeled primer to the DNA template (56%) and to the enzyme (20%), respectively. The lowest efficiency of photocrosslinking was observed for PRII (about 1%).
...
PMID:[Reagents for modification of protein-nucleic acids complexes. II. Site-specific photomodification of DNA-polymerase beta complexes with primers elongated by the dCTP exo-N-substituted arylazido derivatives]. 1144 42

In this paper, we describe the development and use of enzymatic assays to determine intracellular lamivudine triphosphate (3TCTP) and carbovir triphosphate (CBVTP) concentrations in peripheral blood mononuclear cells (PBMCs) from human immunodeficiency virus (HIV)-infected patients. The assays involve inhibition of HIV reverse transcriptase (RT), which normally incorporates radiolabeled deoxynucleoside triphosphates into a synthetic template primer. For the 3TCTP assay, a preincubation procedure was added whereby 3TCTP becomes incorporated before [(3)H]dCTP. At a 1:400 template primer dilution, control product formation was reduced by 88.0% with 0.8 pmol of 3TCTP. Standard 3TCTP inhibition curves were performed using this procedure. For the CBVTP assay, 0.1 pmol of CBVTP inhibited control product formation with and without the use of a preincubation step, so inhibition curves were constructed using both procedures. However, reduced template primer stability with assays using preincubation steps led to a single-incubation procedure being adopted for future studies. The presence of PBMC extracts interfered with the 3TCTP assay. However, this was overcome by the addition of CuSO(4). PBMC extracts did not interfere with the CBVTP assay. Intracellular 3TCTP and CBVTP concentrations were determined in PBMCs from HIV-infected patients over 24 h or greater. Peak concentrations were obtained 6 to 8 h after dosing, and the half-lives of the anabolites suggested the possibility of once-daily dosing. These assays are currently being used for determination of 3TCTP and CBVTP concentrations in clinical studies.
...
PMID:Development of enzymatic assays for quantification of intracellular lamivudine and carbovir triphosphate levels in peripheral blood mononuclear cells from human immunodeficiency virus-infected patients. 1175 Nov 24

Misincorporation at a DNA-carcinogen adduct may contribute to formation of mutations if a polymerase proceeds past the lesion, compromising fidelity, as in the G:C to A:T mutations caused by O(6)-alkylguanine. Replication of primer/templates containing guanine (G), O(6)-methylguanine (O(6)-MeG), or O(6)-benzylguanine (O(6)-BzG) was assessed using T7 DNA polymerase exo(-) (T7(-)) and HIV-1 reverse transcriptase (RT). The steady-state parameters indicated that T7(-) and RT preferentially incorporated dTTP opposite O(6)-MeG and O(6)-BzG. The incorporation efficiencies (k(cat)/K(m)) were less for O(6)-BzG than O(6)-MeG for both dCTP and dTTP insertion. Pre-steady-state analysis indicated that the product formed during the burst phase, i.e., the burst amplitude, differed significantly between the unmodified 24-mer/36-G-mer and the O(6)-alkylG-containing substrates. Extension of the O(6)-BzG-containing duplexes was much more difficult for both polymerases as compared to O(6)-MeG, except when RT easily extended the O(6)-BzG:T base pair. The for binding of dCTP or dTTP to a RT*DNA complex containing O(6)-MeG was 8-fold greater than for dNTP binding to a complex containing unmodified DNA. The for a RT*DNA complex containing O(6)-BzG was 50-fold greater. In conclusion, the bulkier O(6)-BzG is a greater block to polymerization by T7(-) and RT than is O(6)-MeG, but some polymerization does occur with an O(6)-BzG substrate. Pre-steady-state analysis indicates that neither dCTP nor dTTP insertion is strongly preferred during polymerization of O(6)-BzG-containing DNA, unlike the case of O(6)-MeG. These results and others regarding polymerase stalling opposite O(6)-MeG and O(6)-BzG are discussed in the following paper in this issue [Woodside, A. M., and Guengerich, F. P. (2002) Biochemistry 41, 1039-1050].
...
PMID:Effect of the O6 substituent on misincorporation kinetics catalyzed by DNA polymerases at O(6)-methylguanine and O(6)-benzylguanine. 1179 Jan 27

Oxidatively modified deoxynucleotide triphosphates (dN(oxo)TPs) present in nucleotide precursor pools may contribute to retroviral mutagenesis as a result of incorporation and ambiguous base pairing during reverse transcriptase mediated replication. We have examined the incorporation of 5-hydroxy-2'-deoxycytosine triphosphate (5-HO-dCTP) and 2'-deoxyinosine triphosphate (dITP) by HIV-1 reverse transcriptase (HIV-1 RT) on DNA and RNA templates of the same sequence in order to evaluate their mutagenic potential. Significant variations in insertion frequencies at homologous nucleotide positions were observed for each dN(oxo)TP, in general favoring the RNA template. A comparison of steady-state kinetics revealed a 10-fold preference for 5-HO-dCTP incorporation opposite G in RNA. Insertion frequencies for dITP were 2- to 20-fold greater on RNA for every base position examined. One exception to this general trend was observed for the insertion of 5-HO-dCTP by HIV-1 RT opposite A, which favored the DNA template by 4-fold. Deoxyinosine triphosphate was inserted opposite C with an 8-fold higher frequency compared to dGTP in RNA, while on DNA templates, the incorporation frequencies were equivalent. However, incorporation of dITP opposite other bases was characterized by relatively low frequencies. The RNA template bias observed for dN(oxo)TP incorporation is discussed in terms of recent efforts to utilize 5-OH-dCTP as an anti-HIV agent.
...
PMID:Incorporation of oxidatively modified 2'-deoxynucleotide triphosphates by HIV-1 RT on RNA and DNA templates. 1201 86

Human monocytes/macrophages are target cells for HIV-1 infection. As other non-dividing cells, they are characterized by low and imbalanced intracellular dNTP pool levels and an excess of dUTP. The replication of HIV-1 in this cellular context favors misincorporation of uracil residues into viral DNA because of the use of dUTP in place of dCTP. We have previously reported that the host uracil DNA glycosylase enzyme UNG2 is packaged into HIV-1 viral particles via a specific association with the integrase domain of the Gag-Pol precursor. In this study, we investigated whether virion-associated UNG2 plays a role similar to that of its cellular counterpart. We show that the L172A mutation of integrase impaired the packaging of UNG2 into viral particles. Using a primer-template DNA substrate containing G:U mispairs, we demonstrate that wild-type viral lysate has the ability to repair G:U mismatched pairs to G:C matched pairs, in contrast to UNG2-deficient viral lysate. Moreover, no correction of G:T mispairs by wild-type HIV-1 viral lysate was observed, which argues for the specificity of the repair process. We also show that UNG2 physically associates with the viral reverse transcriptase enzyme. Altogether our data indicate for the first time that a uracil repair pathway is specifically associated with HIV-1 viral particles. However, the molecular mechanism of this process remains to be characterized further.
...
PMID:Functional role of HIV-1 virion-associated uracil DNA glycosylase 2 in the correction of G:U mispairs to G:C pairs. 1245 23

This study was aimed to apply an LC-MS-MS method previously developed for intracellular nucleoside reverse transcriptase inhibitors-triphosphate (NRTI-TPs) to the determination of natural deoxyribonucleotides (dNTPs) in human peripheral blood mononuclear cells. The LC-MS-MS method was directly used in assay of dATP and dTTP. Interferences by ribonucleotides (rNTPs) prevented direct application to the two other analytes: dGTP and dCTP. A periodate oxidation procedure was therefore optimized to remove rNTPs from the cell medium in order to quantitate dCTP and dGTP. The determination of the intracellular ratio of NRTI-TP/dNTP in HIV-infected patients now involves use of the same chromatographic system for simultaneous assay of several analytes.
...
PMID:Liquid chromatography-tandem mass spectrometry assays for intracellular deoxyribonucleotide triphosphate competitors of nucleoside antiretrovirals. 1274 19

Commercially available human complementary DNA microarrays were used to compare differential expression in the livers of Atlantic salmon ( Salmo salar) infected with Aeromonas salmonicida and of healthy fish. Complementary DNA probes were prepared from total RNA isolated from livers of control salmon and infected salmon by reverse transcription in the presence of (33)P-dCTP and independently hybridized to human GENE-FILTERS GF211 microarrays. Of the 4131 known genes on the microarray, 241 spots gave clearly detectable signals using labeled RNA from the control salmon liver. Of these, 4 spots were consistently found to have a greater than 2-fold increase in infected salmon compared with controls when using the same pair of filters to generate hybridization data from triplicates. These up-regulated genes were ADP/ATP translocase (AAT2), Na(+)/K(+) ATPase, acyloxyacyl hydrolase (AOAH), and platelet-derived growth factor (PDFG-A). A BlastN search revealed an AAT2 homolog from Atlantic salmon, and a reverse transcriptase polymerase chain reaction assay using primers based on this sequence confirmed its up-regulation (approx. 1.8-fold) during early infection. This work demonstrates the feasibility of using human microarrays to facilitate the discovery of differentially expressed genes in Atlantic salmon, for which no homologous microarrays are available.
...
PMID:Use of human cDNA microarrays for identification of differentially expressed genes in Atlantic salmon liver during Aeromonas salmonicida infection. 1450 54

The Rp-stereoisomer of 5'-(alpha-P-borano)triphosphates of 2'-deoxycytidine (Rp-dCTPalphaB) and 2',3'-dideoxycytidine (Rp-ddCTPalphaB) were synthesized. Their steady-state kinetics of incorporation by ddNTP-resistant enzymes, e.g., MMLV reverse transcriptase (RT) and Taq DNA polymerase, were investigated and compared with incorporation of dCTP and ddCTP. The alpha-boranophosphate substitution in ddCTP results in a 28-fold increase in efficiency of incorporation of the Rp-ddCTPalphaB isomer by MMLV RT, yet has minimal effect on the efficiency of incorporation by Taq DNA polymerase.
...
PMID:Incorporation of (alpha-P-borano)-2',3'-dideoxycytidine 5'-triphosphate into DNA by drug-resistant MMLV reverse transcriptase and Taq DNA polymerase. 1456 87

The use of L(-)SddC [beta-L-2',3'-dideoxy-3'-thiacytidine (lamivudine, 3TC)] for the treatment of Herpes B virus (HBV) infection is hindered by the emergence of drug-resistance associated with the L526M, L550V, and L526M/M550V mutations of the viral DNA polymerase (DP). The interactions of the anti-HBV compounds 2',3'-dideoxy-2',3'-didehydro-beta-L(-)-5-fluorode-oxycytidine and 2'-fluoro-5-methyl-beta-L-arabinofuranosyluracil triphosphate with HBV DP and its L(-)SddC-associated mutants have not been studied. The e antigen-negative variant of HBV associated with the G1896A mutation in the precore region has a high prevalence. Its effect on HBV DP is unclear. Because HBV DNA synthesis occurs in the nucleocapsid, we examined the kinetics of the reverse transcriptase activity from wild-type (wt) and mutated DPs with the wt or G1896A-mutated RNA template in the nucleocapsid. The effects of this template mutation on the activities of these L-nucleoside triphosphates were also examined. Results indicated that these DP mutations increased the Km values of deoxy-NTPs and decreased the efficiencies (Vmax/Km) of DPs. The additional L526M mutation increased the efficiency of the M550V-mutated DP but no more than that of the L526M-mutated DP. The G1896A mutation had impacts on the interactions between different DPs and deoxy-NTPs, except dCTP. It also had different impacts on the actions of the L-nucleoside triphosphates toward DPs. The L526M and M550V mutations caused a greater decrease in the Vmax using the wt RNA template compared with the G1896A-mutated template. The L526M, M550V, and L526M/M550V mutations caused varying degrees of resistance to the different M-nucleoside triphosphates.
...
PMID:Reverse transcriptase activity of hepatitis B virus (HBV) DNA polymerase within core capsid: interaction with deoxynucleoside triphosphates and anti-HBV L-deoxynucleoside analog triphosphates. 1474 82


<< Previous 1 2 3 4 5 6 7 8 9 10 Next >>

---