Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.7.48 (transcriptase)
 9,479 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Some natural and glycon-modified dNTPs with beta,gamma-pyrophosphate substitution at the triphosphate residue were synthesized and studied to evaluate the effect of these modifications on substrate properties of dNTPs in DNA synthesis catalyzed by human placental DNA polymerases alpha and beta, avian myeloblastosis virus reverse transcriptase, and calf thymus terminal deoxynucleotidyl transferase. Reverse transcriptase proved to be the enzyme least specific to such modifications; the substrate activity of beta,gamma-methylenediphosphonate substituted dTTP and 3'-azido-3'-deoxy-dTTP decreased in the following order: CF2 = CHF > CBr2 > CFMe >> CH2. This order is individual for each DNA polymerase. It is interesting to mention that beta,gamma-CBr2 substituted dTTP is neither a substrate nor an inhibitor of DNA polymerase beta. This specificity distinguishes DNA polymerase beta from other DNA polymerases studied.
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PMID:Effect of triphosphate modifications in 2'-deoxynucleoside 5'-triphosphates on their specificity towards various DNA polymerases. 923 75

In this study, human oropharyngeal epidermoid carcinoma KB cells that were resistant to 2,2-difluorodeoxycytidine (dFdCyd) were selected and designated the KB-Gem clone. The KB parental cell line IC50 was 0.3 microM dFdCyd, as compared with the KB-Gem clone IC50 of 32 microM dFdCyd. The KB-Gem clone demonstrated overexpression of ribonucleotide reductase (RR) M2 subunit mRNA (9-fold) and overexpression of M2 protein (2-fold); RR activity was 2.3-fold higher than the KB parental cell line. Both the dATP and dCTP pools of the KB-Gem clone increased 2-fold over the parental cell line, with no change in the dGTP and dTTP pools. Reverse transcriptase-PCR was used to clone the cDNA of deoxycytidine kinase (DCK). Resulting sequences revealed two silent mutations in the KB-Gem clone. The amino acid sequence of the DCK protein and mRNA expression remained unchanged. The KB-Gem clone's DCK enzyme activity was 56% of that of the parental cell line. After the endogenous dNTPs were removed with a G-25 column, no difference was evident between the enzyme activities of the KB-Gem clone and parental cells. Thus, contrary to previous hypotheses, DCK deficiency does not play the primary role in the resistance mechanism of dFdCyd, accepting a secondary role to the overexpression of the target gene, RR, and pool expansion.
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PMID:Overexpression of ribonucleotide reductase as a mechanism of resistance to 2,2-difluorodeoxycytidine in the human KB cancer cell line. 1048 55

We report the crystal structure of the RNA-dependent RNA polymerase of hepatitis C virus, a major human pathogen, to 2.8-A resolution. This enzyme is a key target for developing specific antiviral therapy. The structure of the catalytic domain contains 531 residues folded in the characteristic fingers, palm, and thumb subdomains. The fingers subdomain contains a region, the "fingertips," that shares the same fold with reverse transcriptases. Superposition to the available structures of the latter shows that residues from the palm and fingertips are structurally equivalent. In addition, it shows that the hepatitis C virus polymerase was crystallized in a closed fingers conformation, similar to HIV-1 reverse transcriptase in ternary complex with DNA and dTTP [Huang H., Chopra, R., Verdine, G. L. & Harrison, S. C. (1998) Science 282, 1669-1675]. This superposition reveals the majority of the amino acid residues of the hepatitis C virus enzyme that are likely to be implicated in binding to the replicating RNA molecule and to the incoming NTP. It also suggests a rearrangement of the thumb domain as well as a possible concerted movement of thumb and fingertips during translocation of the RNA template-primer in successive polymerization rounds.
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PMID:Crystal structure of the RNA-dependent RNA polymerase of hepatitis C virus. 1055 68

LALP70 is a novel lysosomal membrane protein belonging to the apyrase protein family. The apyrase protein family comprises enzymes capable of cleaving nucleotide tri- and diphosphates in a calcium- or magnesium-dependent manner, not being altered by P-type, F-type, or V-type NTPase inhibitors. In this study we have cloned and sequenced the human LALP70 gene to determine the genomic structure. The gene is organized in 11 introns and 12 exons covering a genomic region of approximately 16 kilobase pairs. By fluorescence in situ hybridization analysis, the hLALP70 gene was mapped to the human chromosome 8p21.1-p21.3. We further show that there is at least one alternatively spliced variant, hLALP70v, which can be generated via an alternative splice side at the 3'-end of exon 7, leading to a protein variant differing in 8 amino acids (VSFASSQQ). This is the first splice variant that has been described in the apyrase protein family. Reverse transcriptase polymerase chain reaction analysis showed an ubiquitous expression of both variants, with different relative mRNA expression levels in different tissues. Comparison of the enzymatic properties of the splice variants revealed a broader substrate specificity for hLALP70v with CTP, UDP, CDP, GTP, and GDP as preferred substrates, while hLALP70 utilized UTP and TTP preferentially. Furthermore, enzyme activity of hLALP70v was equally dependent on Ca(2+) and Mg(2+), being saturated already at 1 mm concentration. In contrast, hLALP70 enzymatic activity were unsaturated up to 10 mm Ca(2+), while Mg(2+) showed a saturation at already 1 mm concentration with 2-3-fold lower enzymatic activity as observed with Ca(2+). Our data suggest that the presence or absence of the 8-amino acid motif VSFASSQQ provoke differences in substrate specificity and divalent cation dependence of hLALP70/hLALP70v.
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PMID:First apyrase splice variants have different enzymatic properties. 1085 52

Fluorescent nucleic acid hybridization probes traditionally have been generated by enzymatic incorporation of dye-labeled nucleotides, even though incorporation efficiency is low and variable from dye to dye. Alternatively, 5-(3-aminoallyl)-2'-deoxyuridine 5'-triphosphate (aa-dUTP) is enzymatically incorporated to generate amine-modified DNA, which is then chemically labeled with an amine-reactive fluorescent dye. We optimized this latter two-step approach for maximal hybridization signal brightness using DNA probes labeled to varying degrees with different fluorescent dyes. Reverse transcriptase and DNA polymerase 1 efficiently incorporated aa-dUTP into DNA, and adjusting the aa-dUTP:dTTP ratio controlled the degree of substitution. With cDNA probes hybridized to dot blots, probes having approximately eight dyes per 100 bases gave the best sensitivity, irrespective of the dye label. alpha-Satellite probes generated by nick translation and hybridized to human chromosome spreads also showed that probes having approximately eight dyes per 100 bases provided the brightest overall signals. These data demonstrate that this labeling method generates highly sensitive DNA probes that are difficult to obtain by conventional direct incorporation approaches. The technique is inherently consistent and versatile by virtue of the efficient incorporation of primary amines and the reliable chemical labeling reaction.
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PMID:Fluorescent DNA hybridization probe preparation using amine modification and reactive dye coupling. 1474 Apr 93

Hepatitis E virus (HEV) is a causative agent of acute hepatitis, and it is the sole member of the genus Hepevirus in the family Hepeviridae. The open reading frame 1 (ORF1) protein of HEV encodes nonstructural polyprotein with putative domains for methyltransferase, cysteine protease, helicase and RNA-dependent RNA polymerase. It is not yet known whether ORF1 functions as a single protein with multiple domains or is processed to form separate functional units. On the basis of amino acid conserved motifs, HEV helicase has been grouped into helicase superfamily 1 (SF-1). In order to examine the RNA helicase activity of the NTPase/helicase domain of HEV, the region (amino acids 960 to 1204) was cloned and expressed as histidine-tagged protein in Escherichia coli (HEV Hel) and purified. HEV Hel exhibited NTPase and RNA unwinding activities. Enzyme hydrolyzed all rNTPs efficiently, dATP and dCTP with moderate efficiency, while it showed less hydrolysis of dGTP and dTTP. Enzyme showed unwinding of only RNA duplexes with 5' overhangs showing 5'-to-3' polarity. We also expressed and purified two HEV Hel mutants. Helicase mutant I, with substitution in the nucleotide-binding motif I (GKS to GAS), showed 30% ATPase activity. Helicase mutant II, with substitutions in the Mg(2+) binding motif II (DEAP to AAAP), showed 50% ATPase activity. Both mutants completely lost ability to unwind RNA duplexes with 5' overhangs. These findings represent the first report demonstrating NTPase/RNA helicase activity of the helicase domain of HEV ORF1.
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PMID:NTPase and 5' to 3' RNA duplex-unwinding activities of the hepatitis E virus helicase domain. 2007 63

Reverse transcription (RT) is the synthesis of complementary deoxyribonucleic acids (DNA) from single-stranded ribonucleic acid (RNA) templates. This process is catalyzed by the reverse transcriptase enzyme, which is the replicating enzyme of retroviruses. Reverse transcriptase was discovered in 1970, and since then, it has played an instrumental role in the advancement of molecular biology and biotechnology research. In the presence of all four deoxynucleotides (dNTP: dATP, dCTP, dGTP, and dTTP) and under well-defined salt and pH conditions, the reverse transcriptase extends a primer complementary to the RNA to produce a complementary DNA (cDNA) for the RNA template. In this chapter, a simple method of reverse transcription of total cellular RNA into cDNA is described using Superscript II reverse transcriptase (Invitrogen); the resulting cDNA can be used in polymerase chain reaction (PCR).
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PMID:Reverse transcription of the ribonucleic acid: the first step in RT-PCR assay. 2030 Oct 3

Picornaviridae is one of the largest viral families and is composed of 14 genera, six of which include human pathogens. The best known picornaviruses are enteroviruses (including polio, PV, and rhinoviruses), foot-and-mouth disease virus (FMDV), and hepatitis A virus (HAV). Although infections often are mild, certain strains may cause pandemic outbreaks accompanied with meningitis and/or paralysis. Vaccines are available for PV, HAV and FMDV. When the oral vaccines are given to immunocompromised individuals, they may be chronically infected, and remain secretors of vaccine-derived variants of virus for years. There is no effective prophylaxis available for these or other picornaviruses. So far, only the 3C protease from viruses in three genera has been fully characterized as an anti-viral target, whereas the mode of action of compounds targeting other non-structural proteins have remained largely unaddressed. Within the EU-supported FP6 project-VIZIER (Comparative Structural Genomics of Viral Enzymes Involved in Replication), the non-structural proteins were studied to identify conserved binding sites for broadly reactive anti-virals. The putative 2C helicase from echovirus-30 was shown to form ring-shaped hexamers typical for DNA-encoded SF3 helicases, and to possess ATPase activity. Hexamer formation of 2C from enterovirus 76 was in vitro shown to be dependent on the 44 N-terminal residues. Crystal structures of three enterovirus 3C proteases were solved and shown to be similar to those of other picornaviruses. A new binding site of VPg to the bottom of the thumb domain of CV-B3 3D polymerase was identified as a potential target. Broad anti-enterovirus compounds against 2C and 3A proteins were also identified, including thiazolobenzimidazoles (active against 2C) and TTP-8307 (targeting 3A). There is a need for more potent inhibitors against PV and other picornaviruses, which are potential silent reservoirs for re-emerging PV-like disease.
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PMID:Picornavirus non-structural proteins as targets for new anti-virals with broad activity. 2123 2

The human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) converts the viral single-stranded RNA into double-stranded DNA. The inhibition of reverse transcription in the viral life cycle has proven its efficacy as a clinically relevant antiviral target, but the appearance of resistance mutations remains a major cause of treatment failure and stresses the continuous need for new antiviral compounds. In this chapter, we describe an HIV-1 RT scintillation proximity assay (SPA) to identify inhibitors of the RT. The assay uses an RNA/DNA (poly(rA)/oligo(dT)) template/primer bound to SPA beads, which contain scintillant. Reverse transcriptase extends the primer by incorporating [(3)H]dTTP and dTTP, which results in light production by the scintillant in the bead. Compounds that inhibit reverse transcriptase will prevent the incorporation of tritiated dTTP resulting in a reduction of emitted light compared to the untreated controls.
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PMID:Identification of HIV-1 reverse transcriptase inhibitors using a scintillation proximity assay. 2382 Dec 57


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