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 inhibitor captan (N-trichloromethylthio-4-cyclohexen-1,2-dicarboximide) was used to explore the ribonuclease H (RNase H) active site of avian myeloblastosis virus (AMV) reverse transcriptase. Gel permeation chromatography of purified enzyme showed that [14C]captan bound to the alpha subunit in a ratio of 10:1 and to a 32,000 d polypeptide in a ratio of 4:1. Neither the alpha beta nor the beta subunit bound [14C]captan. The binding of 5 of the captan molecules was prevented by preincubating enzyme with polynucleotide. Deoxyguanosine triphosphate (dGTP) protected the enzyme against the binding of 4 captan molecules. Each holoenzyme bound 2 molecules of [3H]dGTP in the absence of, and 1 molecule of [3H]dGTP in the presence of 1 mM captan. Ribonuclease H activity was inhibited when AMV reverse transcriptase was preincubated with 1 mM captan before the degradative reaction was initiated. Preincubation of enzyme with polynucleotide before exposure to captan could partially protect the RNase H activity (61 +/- 2% activity remained). Deoxyguanosine triphosphate also partially protected the RNase H activity from inhibition by captan (75 +/- 9% activity remained). Inhibition of the RNase H activity was completely prevented by preincubating enzyme simultaneously with polynucleotide and dGTP. When separated by glycerol gradients the alpha subunit and alpha beta dimer both exhibited RNase H activity, but only the RNase H activity of the alpha subunit was inhibited by captan. Activity and binding studies revealed that the RNase H and polymerase activities of the alpha subunit are not susceptible to the interaction of captan when this subunit is in the alpha beta dimer form.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Captan binding to avian myeloblastosis virus reverse transcriptase and its effect on RNase H activity. 216 33

3-Methylthymine was synthesized into DNA copolymers and deoxynucleoside triphosphate to study its effect on DNA synthesis by the Klenow fragment of Escherichia coli polymerase I and avian myeloblastosis virus reverse transcriptase. Both polymerases were greatly inhibited by template 3-methylthymine. In response to 3-methylthymine, misincorporation of dTTP increased slightly, but occurred only at low levels consistent with spontaneous misincorporation in vitro. Surprisingly, template 3-methylthymine resulted in a striking decrease in background misincorporation, relative to normal incorporation by the Klenow fragment, of dGTP and, to a lesser extent, of dATP and dCTP. The incorporation of 3-methyl-dTTP into DNA was studied using DNA sequencing technology. The Klenow fragment failed to incorporate 3-methyl-dTTP even at 1 mM. Reverse transcriptase incorporated 3-methyl-dTTP opposite adenine, cytosine, and thymine, but at only about 1/40,000th the efficiency of complementary deoxynucleoside triphosphate incorporation. Furthermore, synthesis generally stalled at sites of 3-methyl-thymine incorporation. From these results, we conclude that damage at the central hydrogen-bonding position of thymine abolishes its base-pairing capabilities during DNA synthesis.
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PMID:DNA damage at thymine N-3 abolishes base-pairing capacity during DNA synthesis. 244 69

The duck hepatitis B virus (DHBV)-associated activities of reverse transcriptase and DNA polymerase and their inhibition in vitro were studied. Replicative complexes (RCs) were isolated from DHBV-infected liver by gel chromatography followed by sucrose gradient centrifugation. The RCs were detected by dot blot hybridization, using radiolabeled cloned DHBV DNA as a probe, and by the incorporation of 32P-TTP in the presence of dATP, dCTP, dGTP, and Mg2+ (endogenous DNA polymerase activity). The endogenous DNA polymerase activity associated with RCs was further studied using exogenous templates: reverse transcriptase and DNA polymerase activities were demonstrated using as substrates 32P-TTP and poly(rA) p(dT)12 or poly(dA) p(dT)12-18, respectively. Both activities were biochemically characterized. Their inhibition by various antiviral agents was studied in vitro: actinomycin D, ara-ATP, aphidicolin, suramin, chloroquin, and phosphonoformate. Among these, suramin, chloroquin, phosphonoformate, and ara-ATP were shown to be potent inhibitors of viral reverse transcriptase and DNA polymerase. Studies are now in progress to establish their antiviral activity in vivo.
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PMID:Duck hepatitis B virus: DNA polymerase and reverse transcriptase activities of replicative complexes isolated from liver and their inhibition in vitro. 245 18

The miscoding properties of a 5-bromodeoxyuridine (dB) containing DNA template during in vitro replication have been investigated. 5-bromodeoxyuridine was introduced site-specifically into the amber 16 codon of a 25-mer oligodeoxynucleotide representing part of the sequence of phi x174am16(+)DNA. The dB containing oligodeoxynucleotide served as a template for in vitro replication by DNA polymerase alpha, DNA polymerase I (Escherichia coli) and AMV reverse transcriptase. The amber 16 revertant assay was used to detect the presence of misincorporated bases in the replication products. For all three DNA polymerases, the presence of dB does not constitute a significant barrier to replication. Errors at the position of dB substitution were found to originate exclusively from dGTP:dB mispairing during in vitro replication thus inducing A-T----G-C transitions. The dGTP:dB mismatches are formed at a 2-4-fold higher frequency as compared to dGTP:T mismatches. Our results indicate that the miscoding potential of dB-substituted DNA templates during replication is only weak at the specific site observed.
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PMID:The fidelity of DNA polymerases during in vitro replication of a template containing 5-bromouracil at a specific site. 246 57

Poly (2'-O-methylcytidylic acid) is recognized as a template in reactions catalyzed by RNA-dependent DNA polymerases in the presence of Mn2+ as divalent cation. We report that kinetic data obtained for dGTP and template under optimal experimental conditions in the reaction catalyzed by reverse transcriptase showed some similarities between the poly (2'-O-methylcytidylic acid)/Mn2+ and polyribocytidylic acid/Mg2+ systems. The reaction was inhibited by the action of N-ethylmaleimide and novobiocin, and to a lesser extent by ethidium bromide and tetramethyl ethidium bromide.
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PMID:Kinetic properties of poly (2'-O-methylcytidylic acid)-directed reverse transcriptase reaction. 246 2

Reverse transcriptase from the simian immunodeficiency virus (SIV) was found to have kinetic behavior similar to that of enzyme from the human immunodeficiency virus (HIV). Michaelis constants for the substrates TTP and dGTP and inhibition constants for the inhibitors 3'-azido-3'-deoxythymidine 5'-triphosphate, 2',3'-dideoxythymidine 5'-triphosphate, and 2'-3'-dideoxyguanosine 5'-triphosphate were obtained for SIV reverse transcriptase and were found to be similar to the corresponding values for HIV reverse transcriptase. Thus, the interaction of SIV reverse transcriptase with nucleotide analogs appears to be indistinguishable from that of the HIV enzyme, suggesting that SIV/simian acquired immunodeficiency syndrome (SAIDS) is a potentially good model of AIDS.
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PMID:Kinetics and inhibition of reverse transcriptase from human and simian immunodeficiency viruses. 246 88

Potential antiviral and antitumour nucleosides, 3'-fluoro-2', 3'-dideoxy-adenosine and -guanosine, have been synthesized by the chemical transglycosylation reaction using 5'-O-acetyl-3'-fluoro-2', 3'-dideoxy-thymidine and -uridine as donors of the carbohydrate fragment and persilylated 6-N-benzoyladenine and 2-N-palmitoylguanine as acceptors, respectively. 5'-Triphosphates of 3'-fluoro-2', 3'-dideoxy-thymidine, -cytidine, -adenosine, and -guanosine (dNTP(3'F] were synthesized and tested as terminators in cell-free system of DNA synthesis catalyzed by RNA-directed DNA polymerase (reverse transcriptase, RT) from the avian myeloblastosis virus (AMV) and E. coli DNA polymerase I (Klenow fragment). A method of estimating relative effectiveness of dNTP(3'F) incorporation into DNA growing chain in comparison with the natural substrates was developed. It is shown that, in case of AMV-RT, dATP(3'F), dCTP(3'F) incorporate 14 times less efficiently than dATP and dCTP respectively, and dTTP(3'F) 3 times less effectively than the corresponding natural substrates, whereas dGTP (3'F) is as efficient as dGTP. With E. coli DNA polymerase I (Klenow fragment) dATP (3'F) and dCTP(3'F) are ca. 100 times less efficient, and dTTP(3'F) and dGTP(3'F) are ca. 50 times less efficient than the respective natural substrates.
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PMID:[Synthesis of 2',3',-dideoxy-3'-fluoradenosine and -guanosine, their 5'-triphosphates and a study of 2',3'-dideoxy-3'-fluoronucleoside- 5'-triphosphates as substrates for DNA-polymerases]. 267 51

A number of nucleoside 5'-triphosphate analogs were tested with Escherichia coli DNA polymerase I and Klenow fragment of the enzyme, bacteriophage T4 DNA polymerase and calf thymus DNA polymerase alpha. It was shown that 3'-amino-2',3'-dideoxynucleoside 5'-triphosphates as well as a number of 3'-derivatives of dTTP(3'NH2) are able to terminate DNA synthesis catalyzed by each enzyme if the reaction is performed in the absence of natural substrates. ddNTP and dNTP(3'F) were found to be inactive with DNA polymerase alpha only, but araNTP(3'NH2) was inactive with E. coli DNA polymerase I. dTTP(3'N3), dGTP(3'N'3), dCTP(3'N3), araNTP(3'N3) and (alpha-thio)dTTP(3'F) were unable to inhibit any of the above-mentioned DNA polymerases, in contrast to reverse transcriptase, accessible to the most nucleotide analogs tested.
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PMID:Nucleoside 5'-triphosphates with modified sugars as substrates for DNA polymerases. 302 Dec 25

Incorporation of 2'-deoxyribonucleotide 5'-triphosphate derivatives, chemically modified both in the base and at 3'-position, into DNA by four different DNA polymerases was investigated. It is shown that 3'-azido- and 3'-amino-2',3'-dideoxy-(E)-5-(2-bromovinyl)-uridine 5'-triphosphates effectively terminate DNA synthesis catalyzed by E. coli DNA polymerase I, rat liver DNA polymerase beta, and AMV reverse transcriptase. Calf thymus DNA polymerase alpha incorporates only the 3'-amino derivative. DNA polymerases I and beta catalyse DNA synthesis in the presence of beta-D-(2'-deoxyribofuranosyl)-1-benzimidazol 5'-triphosphate, inserting the corresponding monophosphate in place of -dGTP, whereas 3'-substituted analogues of this compound were inactive in the reactions.
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PMID:[Analogs of deoxyribonucleoside-5'-triphosphates modified in the base and sugar moieties: substrate properties in DNA biosynthesis in cell-free systems]. 343 59

Human thyroglobulin mRNA was isolated from Graves' goitres by size selection of total poly(A)-rich RNA in a sucrose gradient. It sedimented at 33 S, as in other mammalian species, and showed a single component of approximately 8500 bases by gel electrophoresis. cDNA was synthesized from the 33-S RNA by using reverse transcriptase in the presence of human placenta ribonuclease inhibitor and in conditions allowing the formation of long transcripts. The latter was made double-stranded using reverse transcriptase and blunt-ended with nuclease S1. After tailing with dCTP and terminal transferase, the double-stranded cDNA was annealed to pBR322 DNA that had been cleaved at the endonuclease PstI site and tailed with dGTP. The resulting plasmids were used to transform Escherichia coli C600 cells and four cloned recombinants were selected. Each plasmid DNA was shown to contain a sequence complementary to human thyroglobulin mRNA by hybridization with a labeled 33-S mRNA, visualization of cDNA . mRNA hybrids by electron microscopy and filter hybridization selection of mRNA directing the synthesis of immunologically related thyroglobulin peptides in the reticulocyte lysate. The four inserted DNA sequences were 1400 - 1800 base pairs long, two of them showing an homologous sequence of 1100 base pairs. Together, the four cloned DNA fragments represented 63% of the 8500 bases of human thyroglobulin mRNA.
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PMID:Cloning of four DNA fragments complementary to human thyroglobulin messenger RNA. 617 25


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