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:2.7.7.7 (DNA polymerase)
17,007 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Using BspMI cassette vectors, we have constructed a series of mutations in human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) that cause specific amino acid substitutions within the polymerase domain. The RNA-dependent DNA polymerase, DNA-dependent DNA polymerase, and RNase H activities of the mutant RTs were assayed. The elucidation of the structure of HIV-1 RT makes it possible to determine the locations of specific mutations in the three-dimensional structure of HIV-1 RT [E. Arnold, A. Jacobo-Molina, R. G. Nanni, R. L. Williams, X. Lu, J. Ding, A. D. Clark, Jr., A. Zhang, A. L. Ferris, P. Clark, A. Hizi, and S. H. Hughes, Nature (London) 357:85-89, 1992; L. A. Kohlstaedt, J. Wang, J. M. Friedman, P. A. Rice, and T. A. Steitz, Science 256:1783-1790, 1992]. The mutations described in this report are between amino acids 25 and 81, within the "fingers" domain of RT (Kohlstaedt et al., Science 256:1783-1790, 1992). It has been suggested that this domain may play a role in positioning the template. Although the fingers domain does not contain the active site for polymerization, several of the mutations within this domain disrupt polymerase activity without significantly affecting RNase H activity.
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PMID:Mutational analysis of the fingers domain of human immunodeficiency virus type 1 reverse transcriptase. 127 5

beta-L-3'-Deoxythymidine 5'-triphosphate (L-ddTTP) and beta-L-3'-deoxy-2',3'-didehydrothymidine 5'-triphosphate (L-d4TTP) were substrates for human immunodeficiency virus reverse transcriptase, Escherichia coli DNA polymerase I (Klenow), and Sequenase (modified T7 DNA polymerase). The beta-D- and beta-L-enantiomers of 5-methyluridine 5'-triphosphate (rTTP) were inhibitors but not substrates of reverse transcriptase. The steady-state Km values for L-ddTTP and L-d4TTP, with all three enzymes, were 12-70-fold larger than the Km values for the corresponding D-enantiomers. The Km value of reverse transcriptase for L-ddTTP was 50-fold larger than that for D-ddTTP because the Kd for L-ddTTP was 5-fold larger than that for D-ddTTP, and the first-order rate constant for incorporation of L-ddTMP into the template-primer was 10% that of the D-enantiomer. The D- and L-enantiomers had kcat values with reverse transcriptase and Sequenase that were similar to kcat for the natural substrate, thymidine 5'-triphosphate (dTTP). Thus, the rate determining step appeared to be dissociation of the enzyme-chain-terminated template-primer complex. In contrast, kcat values for the L-enantiomers with Klenow were only 0.1% that of dTTP, and the kcat values for the D-enantiomers were 15% the kcat for dTTP. The reduced kcat values were due to a change in rate determining step from dissociation of the Klenow-chain-terminated template-primer complex to an earlier step in the reaction mechanism, presumably catalysis. Thus, these DNA polymerases did not stereospecifically recognize D-nucleoside 5'-triphosphate analogs as substrates.
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PMID:Beta-L-thymidine 5'-triphosphate analogs as DNA polymerase substrates. 128 Nov 53

Reverse transcriptase (RT) was first discovered as an essential catalyst in the biological cycle of retroviruses. However, in the past years evidence has accumulated showing that RTs are involved in a surprisingly large number of RNA-mediated transpositional events that include both viral and nonviral genetic entities. Although it is probable that some RT-bearing genetic elements like the different types of AIDS viruses and the mammalian LINE family have arisen in recent geological times, the possibility that reverse transcription first took place in the early Archean is supported by (1) the hypothesis that RNA preceded DNA as cellular genetic material; (2) the existence of homologous regions of the subunit tau of the E. coli DNA polymerase III with the simian immunodeficiency virus RT, the hepatitis B virus RT, and the beta' subunit of the E. coli RNA polymerase (McHenry et al. 1988); (3) the presence of several conserved motifs, including a 14-amino-acid segment that consists of an Asp-Asp pair flanked by hydrophobic amino acids, which are found in all RTs and in most cellular and viral RNA polymerases. However, whether extant RTs descend from the primitive polymerase involved in the RNA-to-DNA transition remains unproven. Substrate specificity of the AMV and HIV-1 RTs can be modified in the presence of Mn2+, a cation which allows them to add ribonucleotides to an oligo (dG) primer in a template-dependent reaction. This change in specificity is comparable to that observed under similar conditions in other nucleic acid polymerases. This experimentally induced change in RT substrate specificity may explain previous observations on the misincorporation of ribonucleotides by the Maloney murine sarcoma virus RT in the minus and plus DNA of this retrovirus (Chen and Temin 1980). Our results also suggest that HIV-infected macrophages and T-cell cells may contain mixed polynucleotides containing both ribo- and deoxyribonucleotides. The evolutionary significance of these changes in substrate specificities of nucleic acid polymerases is also discussed.
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PMID:On the early emergence of reverse transcription: theoretical basis and experimental evidence. 128 61

An aqueous extract of Phyllanthus niruri (Euphorbiaceae) inhibited human immunodeficiency virus type-1 reverse transcriptase (HIV-1-RT). The inhibitor against HIV-1-RT in this plant was purified by combination of three column chromatographies, Sephadex LH-20, cellulose, and reverse-phase high-performance liquid chromatography. The inhibitor was then identified by nuclear magnetic resonance (NMR) spectra as repandusinic acid A monosodium salt (RA) which was originally isolated from Mallotus repandus. The 50% inhibitory doses (ID50) of RA on HIV-1-RT and DNA polymerase alpha (from HeLa cells) were 0.05 microM and 0.6 microM, respectively, representing approximately a 10-fold more sensitivity of HIV-1-RT compared with DNA polymerase alpha. RA was shown to be a competitive inhibitor with respect to the template-primer while it was a noncompetitive inhibitor with respect to the substrate. RA as low as 10.1 microM inhibited HIV-1-induced cytopathogenicity in MT-4 cells. In addition, 4.5 microM of RA inhibited HIV-1-induced giant cell formation of SUP-T1 approximately 50%. RA (2.5 microM) inhibited up to 90% of HIV-1 specific p24 antigen production in a Clone H9 cell system.
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PMID:HIV-1 reverse transcriptase inhibitor from Phyllanthus niruri. 128 10

Catechin derivatives including (-)-epicatechin gallate (ECG), (-)-epigallocatechin gallate (EGCG), (-)-epigallocatechin (EGC) and green tea extract (GTE) were found to inhibit the activities of cloned human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT), duck hepatitis B virus replication complexes reverse transcriptase (DHBV RCs RT), herpes simplex virus 1 DNA polymerase (HSV-1 DNAP) and cow thymus DNA polymerase alpha (CT DNAP alpha). EGCG and ECG were shown to be very potent inhibitors of HIV-1 RT. According to the IC50 values for HIV-1 RT, these compounds can be ordered as EGCG 0.0066 mumol/L > ECG 0.084 mumol/L > GTE 0.1 microgram/ml > EGC 7.2 mumol/L. DHBV RCs RT was the least sensitive to these compounds. Kinetic study showed that EGCG exerts a mixed inhibition with respect to external template inducer poly (rA).oligo (dT) 12-18 and a noncompetitive inhibition with respect to substrate dTTP for HIV-1 RT. Bovine serum albumin significantly reduced the inhibitory effects of catechin analogues and GTE on HIV-1 RT. In tissue culture GTE inhibited the cytopathic effect of coxsackie B3 virus, but did not inhibit the cytopathic effects of HSV-1, HSV-2, influenza A or influenza B viruses.
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PMID:[The inhibitory effects of catechin derivatives on the activities of human immunodeficiency virus reverse transcriptase and DNA polymerases]. 128 89

A traditional Kampo drug, Sho-saiko-to, composed of several herb extracts, differentially inhibited the activities of reverse transcriptase and human cellular DNA polymerase alpha and beta. Reverse transcriptases from murine leukemia virus and human immunodeficiency virus were inhibited by over 80% and 50%, respectively, in the presence of 100 micrograms/ml Sho-saiko-to, whereas DNA polymerase alpha was much less sensitive to inhibition by this drug than were the reverse transcriptases. DNA polymerase gamma was not inhibited by this drug at concentrations of up to 500 micrograms/ml. Only DNA polymerase beta was moderately inhibited by Sho-saiko-to. Thus, it has been shown that the inhibition by Sho-saiko-to is relatively specific for reverse transcriptase and that the drug contains as yet unidentified inhibitory substance(s) for reverse transcriptase.
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PMID:Differential inhibition of the activities of reverse transcriptase and various cellular DNA polymerases by a traditional Kampo drug, sho-saiko-to. 128 36

2',3'-Dideoxy-3'-thiacytidine (+/-)-SddC) was found to have potent activity against human hepatitis B virus as well as human immunodeficiency viruses in culture. The (-)form ((-)-SddC) which is resistant to deoxycytidine deaminase was found to be the more active antiviral stereoisomer than the (+)-form ((+)-SddC). The (+)-SddC is susceptible to deamination by deoxycytidine deaminase and is 25- and 12-fold more toxic than (-)-SddC in CEM cells in terms of anti-cell growth and anti-mitochondrial DNA synthesis, respectively. Similar results were obtained using a mixture of their 5-fluoro analogs ((+/-)-FSddC). Unlike 2',3'-dideoxycytidine, which is a potent inhibitor of mitochondrial DNA synthesis and results in such delayed toxicity as peripheral neuropathy with long term usage, (-)-SddC does not affect mitochondrial DNA synthesis. The (-)form is phosphorylated to (-)-SddCMP and is subsequently converted to (-)-SddCDP and (-)-SddCTP. One additional major metabolite which has been tentatively assigned the name "(-)-SddCMP sialate" was also identified. No significant difference in terms of the profiles of the metabolites was found between 4 and 24 h. There is an appreciable amount of (-)-SddCTP detectable 24 h after removal of the drug. (-)-SddCTP was also found to be approximately 3-fold more potent than (+)-SddCTP in inhibiting human hepatitis B virus DNA polymerase. This is the first nucleoside analog with the unnatural sugar configuration demonstrated to have antiviral activity.
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PMID:Deoxycytidine deaminase-resistant stereoisomer is the active form of (+/-)-2',3'-dideoxy-3'-thiacytidine in the inhibition of hepatitis B virus replication. 132 Nov 32

2',3'-Dideoxy-3'-thiacytidine (cis-(+/-)-SddC) was found to have potent activity against hepatitis B virus and human immunodeficiency viruses in culture. Recent studies by us identified (-)-SddC as the stereoisomer responsible for the antiviral effect and showed that the cytotoxicity was mainly caused by (+)-SddC. Metabolism studies showed that these drugs were converted to their monophosphates, diphosphates, and triphosphates. The enzyme responsible for the formation of monophosphates was identified to be cytoplasmic deoxycytidine kinase in CEM cells. Uptake studies showed that the intracellular concentration of (-)-SddC and its metabolites was approximately 5-fold higher than that of (+)-SddC metabolites. (-)-SddCTP was more potent than (+)-SddCTP in inhibiting hepatitis B virus replication; (+)- and (-)-SddCTP exhibited minimal inhibition on polymerases alpha and delta, more inhibition on beta, and strong inhibition on gamma. In all cases, (+)-SddCTP was found to be more inhibitory than (-)-SddCTP to all four polymerases. (+)-SddCMP competed with dCTP for incorporation into DNA by DNA polymerase gamma and beta and served as a chain terminator; however, similar incorporation was not detected using other polymerases. The selective inhibition of DNA synthesis in isolated mitochondria by (+)- and (-)-SddCTP suggests a stereospecificity on the mitochondrial uptake of deoxynucleoside triphosphates.
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PMID:Biochemical pharmacology of (+)- and (-)-2',3'-dideoxy-3'-thiacytidine as anti-hepatitis B virus agents. 133 Oct 54

Polymerase chain reaction (PCR) was prospectively performed with cerebrospinal fluid (CSF) from 51 patients whose CSF was available for analysis and was submitted for viral culture and/or herpes simplex virus (HSV) serology and 20 patients whose CSF was submitted exclusively to the Clinical Biochemistry Laboratory. Primers were used that flanked a 92 bp segment of the HSV DNA polymerase gene (35 cycles). Amplified products were electrophoresed on agarose gel, blotted onto nylon membrane, and probed with a 32P-labelled sequence internal to the primers. For nested PCR, 1 microliter of PCR product was amplified for an additional 35 cycles before electrophoresis and Southern blot analysis. Review of the clinical records revealed that 15 patients had central nervous system (CNS) infections. Specific HSV DNA sequences were detected in CSF specimens of three of the individuals [PCR(2), nested PCR(1)]. Two of these patients had disseminated HSV infection including encephalitis and one patient had aseptic meningitis. The diagnoses of the 12 patients with CNS infection who did not have HSV DNA detected in CSF included encephalitis [varicella-zoster virus (1), cytomegalovirus (1), Mycoplasma pneumoniae (1)], meningitis [Neisseria meningitidis (1), Coccidioides immitis (1), Enterovirus (1), aseptic meningitis (1)], varicella-zoster radiculitis (2), human immunodeficiency virus dementia (2), and transverse myelitis due to Epstein-Barr virus (1). Importantly, HSV DNA was also not detected in the CSF of the 36 patients who did not have CNS infection and 20 samples submitted exclusively to the Clinical Biochemistry Laboratory. Our findings demonstrate the utility of PCR as a rapid, non-invasive method for the routine laboratory diagnosis of CNS infection due to HSV.
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PMID:A prospective study of the polymerase chain reaction for detection of herpes simplex virus in cerebrospinal fluid submitted to the clinical virology laboratory. 133 47

A synthetic RNA oligonucleotide (15-mer) corresponding to the 3' end of the lysine tRNA primer was hybridized to single-stranded DNA containing the human immunodeficiency virus type 1 (HIV-1) primer-binding site and extended with a DNA polymerase. The resulting structures were used to study primer removal by the RNase H activity of HIV-1 reverse transcriptase. The initial cleavage event removes the RNA primer as a 14-mer and leaves a single ribonucleotide A residue bound to the 5' end of the DNA strand. This result explains the observation by several groups that HIV-1 circle junctions contain 4 bp that are not present in the integrated provirus instead of the predicted 3 bp. Subsequent cleavage events occur at other sites internal to the RNA molecule, and the ribonucleotide A residue on the end of the DNA strand is ultimately removed. Therefore, the biologically relevant cleavage that produces the 14-mer reflects the kinetics of the reaction as well as a specificity for nucleic acid sequence. When the RNA oligonucleotide alone was hybridized to the primer-binding site and tested as a substrate for HIV-1 RNase H, the cleavage pattern near the 3' end of the RNA was altered.
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PMID:Incomplete removal of the RNA primer for minus-strand DNA synthesis by human immunodeficiency virus type 1 reverse transcriptase. 137 87


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