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 X-ray crystal structure at 2.0 A resolution of a DNA molecule complexed with the N-terminal fragment of Moloney murine leukemia virus reverse transcriptase (MMLV RT) has been determined. This method allows the study of nucleic acids in a unique and largely unfettered environment without the complicated lattice interactions typically observed in DNA-only crystal structures. Molecular-replacement phasing using only the protein provided readily interpretable electron density with no model bias for the DNA. The asymmetric unit of the structure consists of the protein molecule bound to the blunt end of a DNA 6/10-mer, which is composed of a six-base strand (5'-GTCGTC-3') and a ten-base strand (3'-CAGCAGGGCA-5'), resulting in a six-base-pair duplex with a four-base single-stranded overhang. In the crystal structure, the bases of the overhang reciprocally pair to yield a doubly nicked pseudo-hexadecamer primarily B-form DNA molecule. The pairing between the single strands gives two standard (G-C) Watson-Crick pairs and two G(anti)-A(anti) mispairs. The mispairs reside in a G-C-rich environment and the three consecutive guanines on the 10-mer impart interesting structural features to the pseudo-hexadecamer, such as the preference for a guanine stack, stretching the C-G base pairs flanking the mispair to the point of loss of intra-base-pair hydrogen bonding. The DNA was designed for the purpose of comparison with a previous structure, which was determined in the same crystal lattice. In all of the authors' previous fragment-DNA complexes, the nucleotide at the blunt-ended 3'-hydroxyl was a purine. Consistent with the proposed mechanistic role of interactions with the 3'-hydroxyl in processive DNA synthesis by RT, it was found that a pyrimidine at this position in the DNA makes indentical interactions with the strictly conserved Gly191 and the main chain of Leu115 of MMLV RT.
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PMID:Structure of a pseudo-16-mer DNA with stacked guanines and two G-A mispairs complexed with the N-terminal fragment of Moloney murine leukemia virus reverse transcriptase. 1152 15

A growing concern in the pursuit of new therapies for HIV-1 infection is the potential for the virus to develop drug resistance. With the advent of modern antiretroviral therapy and the common use of combined modalities, it is difficult to identify in the clinic the mutations associated with a specific drug. In general, drug selection of mutants using a relevant cell system, such as primary human lymphocytes, is a good prognosticator of what will happen in humans. In this study, HIV-infected human peripheral blood mononuclear cells were exposed, at a concentration of 1- to 10-fold the median effective antiviral concentration, to the nucleosides (-)-beta-2',3'-dideoxy-3'-thia-5-fluorocytidine [(-)-FTC] (-)-beta-2',3'-dideoxy-3'-thiacytidine (3TC), 3'-azido-2',3'-dideoxyuridine (CS-87, AZDU), 3'-azido-2',3'-dideoxy-5-methylcytidine (CS-92, AZMC), 2',3'-didehydro-3'-deoxythymidine (d4T), beta-L-2',3'-didehydro-2',3'-dideoxy-5-fluorocytidine (beta-L-D4FC), beta-L-2',3'-dideoxyadenine SATE[beta-L-ddAMP-bis(tbutylSATE)], beta-L-5-fluoro-2',3'-dideoxycytidine (L-FddC), and the protease inhibitors nelfinavir and amprenavir (VX-478). Virus from the culture supernatant was amplified by PCR and analysed by both HIV-1 reverse transcriptase and protease line probe assay. All the L-nucleoside analogues tested selected for the V184 mutation, including the L-pyrimidine nucleosides 3TC (-)-FTC, beta- L-FddC, beta-L-D4FC and the beta-L-purine nucleoside. beta-L-D4FC also selected for K/R65 in addition to V184, indicating that these two mutations are linked and compatible in vitro. No pattern of mutations leading to resistance or reduced susceptibility was discerned with d4T. Rapid genotyping analysis revealed the different kinetics and mutations obtained by in vitro selection in HIV-infected cells exposed to nucleoside analogues and protease inhibitors.
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PMID:Early detection of mixed mutations selected by antiretroviral agents in HIV-infected primary human lymphocytes. 1159 90

Functional analyses of a number of hydrolase gene promoters, induced by gibberellin (GA) in aleurone cells following germination, have identified a GA-responsive complex as a tripartite element containing a pyrimidine box motif 5'-CCTTTT-3'. We describe here that BPBF, a barley (Hordeum vulgare) transcription factor of the DOF (DNA-Binding with One Finger) class, previously shown to be an activator of reserve protein encoding genes during development, also has a role in the control of hydrolase genes following seed germination. Northern-blot, reverse transcriptase-polymerase chain reaction, and in situ hybridization analyses evidenced that the transcripts of the BPBF-encoding gene (Pbf), besides being present during endosperm development, are also expressed in aleurone cells of germinated seeds where they are induced by GA, an effect counteracted by abscisic acid. Electrophoretic mobility shift assays have shown that the BPBF protein binds specifically to the pyrimidine box motif in vitro within the different sequence contexts that naturally occur in the promoters of genes encoding a cathepsin B-like protease (Al21) and a low-isoelectric point alpha-amylase (Amy2/32b), both induced in the aleurone layers in response to GA. In transient expression experiments, BPBF repressed transcription of the Al21 promoter in GA-treated barley aleurone layers and reverted the GAMYB-mediated activation of this protease promoter.
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PMID:A role for the DOF transcription factor BPBF in the regulation of gibberellin-responsive genes in barley aleurone. 1222 91

While comparing gene expression in the pathogenic organism Entamoeba histolytica and the closely related but nonpathogenic species Entamoeba dispar, we discovered that the E. histolytica abundant polyadenylated transcript 2 (ehapt2) and corresponding genomic copies are absent in E. dispar. Although polyadenylated, ehapt2 does not contain any overt open reading frame. Southern blot and sequence analyses revealed that about 500 copies of ehapt2 genomic elements were present in each cell and that the copies were distributed throughout the ameba genome. The various ehapt2 elements are regularly located in the vicinity of protein-encoding genes, downstream of pyrimidine-rich sequence stretches (40 to 125 bp; CT content, 79.2 to 85.5%), and are flanked by duplicated target sites of variable length. Target site duplications were obviously generated during integration of ehapt2 into the E. histolytica genome as one copy of the flanking repeat and the complete ehapt2 element are specifically absent in orthologous E. dispar genomic sequences. ehapt2 shares 3' sequences with EhRLE, a recently identified non-long-terminal-repeat (non-LTR) retrotransposon-like element of E. histolytica, which contains a conceptual open reading frame for reverse transcriptase. Thus, ehapt2 has all of the properties of nonautonomous non-LTR retrotransposons. A comparison of various E. histolytica isolates suggested that transposition of ehapt2 takes place at a very low frequency as the genomic localization of ehapt2 elements was found to be well conserved. A mobile element such as ehapt2 could be a suitable mechanism to explain the infrequent and late transition of E. histolytica from a harmless gut commensal to an invasive pathogen.
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PMID:The abundant polyadenylated transcript 2 DNA sequence of the pathogenic protozoan parasite Entamoeba histolytica represents a nonautonomous non-long-terminal-repeat retrotransposon-like element which is absent in the closely related nonpathogenic species Entamoeba dispar. 1243 55

The tumor suppressor protein p53 plays an important role in maintenance of the genomic integrity of cells. p53 possesses an intrinsic 3'-->5' exonuclease activity. p53 was found in the nucleus and in the cytoplasm of the cell. In order to evaluate the subcellular location and extent of p53-associated 3'--> 5' exonuclease activity, we established an in vitro experimental system of cell lines with different nuclear/cytoplasmic distribution of p53. Nuclear and cytoplasmic extracts obtained from LCC2 cells (expressing a high level of cytoplasmic wild-type p53), MCF-7 cells (expressing a high level of wild-type nuclear p53), MDA cells (expressing mutant p53) and H1299 cells (p53-null) were subjected to the analysis of exonuclease activity. Interestingly, 3'-->5' exonuclease was predominantly cytoplasmic; the nuclear extracts derived from all cell lines tested, exerted a low level of exonuclease activity. Cytoplasmic extracts of LCC2 cells, with a high level of wild-type p53, showed an enhanced exonuclease activity in comparison to those expressing either a low level of wild-type p53 (in MCF-7 cells) or the mutant p53 (in MDA cells). Evidence that exonuclease function detected in cytoplasmic extracts is attributed to the p53 is supported by several facts: First, this activity closely parallels with levels and status of endogenous cytoplasmic p53. Second, immunoprecipitation of p53 from cytoplasmic extracts of LCC2 cells markedly reduced the exonuclease activity. Third, the observed 3'-->5' exonuclease in cytoplasmic fraction of LCC2 cells displays identical biochemical properties characteristic of recombinant wild-type p53. The biochemical functions include: (a) substrate specificity; exonuclease hydrolyzes single-stranded DNA in preference to double-stranded DNA and RNA/DNA template-primers, (b) efficient excision of 3'-terminal mispairs from DNA/DNA and RNA/DNA substrates, (c) the preferential excision of purine-purine mispairs over purine-pyrimidine mispairs and (d) functional interaction with exonuclease-deficient DNA polymerase, for example, murine leukemia virus reverse transcriptase (representing a relatively low fidelity enzyme), thus enhancing the fidelity of DNA synthesis by excision of mismatched nucleotides from the nascent DNA strand. Taken together, the data demonstrate that wild-type p53 in cytoplasm, in its noninduced state, is functional; it displays intrinsic 3'-->5' exonuclease activity. The possible role of p53-associated 3'-->5' exonuclease activity in DNA repair in nucleus and cytoplasm is discussed.
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PMID:p53-associated 3'-->5' exonuclease activity in nuclear and cytoplasmic compartments of cells. 1252 92

Thymidine phosphorylase (dThdPase) is identical to platelet-derived endothelial cell growth factor, and has an angiogenic activity in experimental models and human solid tumors. A critical step in the de novo pathway of DNA synthesis is the production of the pyrimidine nucleotide dTMP from dump and this reaction is catalyzed by thymidylate synthase (TS). Both dThdPase and TS levels seemed to be related to response to 5-fluorouracil (5-FU) therapy in different types of human solid tumors. The present study evaluated dThdPase and TS expression levels by immunohistochemistry and reverse transcriptase-polymerase chain reaction (RT-PCR), using a same set of 39 breast carcinoma tissues for both methods. An inverted-relationship in the expression levels of TS and dThdPase was observed. Further, immunohistochemical analysis may be a better tool than analysis by RT-PCR in detection of dThdPase and TS, because of both dThdPase and TS expression in cells besides carcinoma cells. These imply that immunohistochemical analysis of dThdPase and TS is available for selection of patients who will be received 5-FU based chemotherapy.
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PMID:Expression of thymidylate synthase and thymidine phosphorylase in human breast carcinoma: implication for method to detect expression of these molecules in clinic. 1253 82

beta-l-2',3'-Didehydro-2',3'-dideoxy-2'-fluoro-4'-thionucleosides (beta-l-2'-F-4'-S-d4Ns) have been synthesized and evaluated against HIV-1 in primary human lymphocytes. The key intermediate 8, which was prepared from 2,3-O-isopropylidene-l-glyceraldehyde 1 in 13 steps, was condensed with various pyrimidine and purine bases followed by elimination and deprotection to give the target compounds, beta-l-2'-F-4'-S-d4Ns (17-20 and 27-30). The antiviral activity of the newly synthesized compounds was evaluated against HIV-1 in human peripheral blood mononuclear (PBM) cells, among which the cytosine 17, 5-fluorocytosine 18, and adenine 27 derivatives showed potent anti-HIV activities (EC(50) = 0.12, 0.15, and 1.74 microM, respectively) without significant cytotoxicity up to 100 microM in human PBM, CEM, and Vero cells. The cytosine derivative 17 (beta-l-2'-F-4'-S-d4C), however, showed cross-resistance to a 3TC-resistant variant (HIV-1(M184V)). Molecular modeling studies suggest that the pattern of antiviral activity, similar to that of beta-l-2'-F-d4N, stemmed from their conformational and structural similarities. The isosteric substitution of sulfur for 4'-oxygen was well tolerated in the catalytic site of HIV-1 reverse transcriptase in the wild-type virus. However, the steric hindrance between the sugar moiety of the unnatural l-nucleoside and the side chains of Val184 of M184V RT in 3TC-resistant mutant HIV strains destabilizes the RT-nucleoside triphosphate complex, which causes the cross-resistance to 3TC (M184V mutant).
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PMID:Synthesis, anti-HIV activity, and molecular mechanism of drug resistance of L-2',3'-didehydro-2',3'-dideoxy-2'-fluoro-4'-thionucleosides. 1254 Feb 38

Hybridization properties of oligodeoxyxylonucleotides (OXNs) built from pyrimidine monomers with an inverted 3'-OH group of the furanose have been studied using the gel mobility shift, UV melting and circular dichroism (CD) spectroscopy methods. Pyrimidine OXNs form triple helices with complementary purine RNA in which one OXN is parallel and another is antiparallel with respect to the RNA target. Surprisingly, no duplex formation between the pyrimidine OXNs and purine RNAs is detected. The modified triplexes are stable at pH 7. Their thermal stability depends on the number of C(G-C) triplets and, for G-rich RNA sequences, it is comparable with the stability of native DNA-RNA duplexes. The CD spectra of triplexes formed by OXNs with purine RNA targets are similar to spectra of A-type helices. A pyrimidine OXN having a clamp structure efficiently inhibits reverse transcription of murine pim-1 mRNA in vitro mediated by the Mo-MuLV reverse transcriptase.
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PMID:Formation of stable triplexes between purine RNA and pyrimidine oligodeoxyxylonucleotides. 1285 44

Beta-D and beta-L-enantiomers of 2',3'-dideoxycytidine analogues are potent chain-terminators and antimetabolites for viral and cellular replication. Seemingly small modifications markedly alter their antiviral and toxicity patterns. This review discusses previously published and recently obtained data on the effects of 5- and 2'-fluorine substitution on the pre-steady state incorporation of 2'-deoxycytidine-5'-monophosphate analogues by HIV-1 reverse transcriptase (RT) in light of their biological activity. The addition of fluorine at the 5-position of the pyrimidine ring altered the kinetic parameters for all nucleotides tested. Only the 5-fluorine substitution of the clinically relevant nucleosides (-)-beta-L-2',3'-dideoxy-3'-thia-5-fluorocytidine (L-FTC, Emtriva), and (+)-beta-D-2',3'-didehydro-2',3'-dideoxy-5-fluorocytidine (D-D4FC, Reverset), caused a higher overall efficiency of nucleotide incorporation during both DNA- and RNA-directed synthesis. Enhanced incorporation by RT may in part explain the potency of these nucleosides against HIV-1. In other cases, a lack of correlation between RT incorporation in enzymatic assays and antiviral activity in cell culture illustrates the importance of other cellular factors in defining antiviral potency. The substitution of fluorine at the 2' position of the deoxyribose ring negatively affects incorporation by RT indicating the steric gate of RT can detect electrostatic perturbations. Intriguing results pertaining to drug resistance have led to a better understanding of HIV-1 RT resistance mechanisms. These insights serve as a basis for understanding the mechanism of action for nucleoside analogues and, coupled with studies on other key enzymes, may lead to the more effective use of fluorine to enhance the potency and selectivity of antiviral agents.
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PMID:Probing the mechanistic consequences of 5-fluorine substitution on cytidine nucleotide analogue incorporation by HIV-1 reverse transcriptase. 1452 28

To decrease the toxicity of potent anti-HIV nucleosides 3'-azido-2',3'-dideoxythymidine (AZT) and 2',3'-dideoxy-3'-fluorothymidine (3'-FddThd, FLT), their new analogues, 3'-azido-2',3'-dideoxy-5-hydroxymethyluridine (3'-Az5HmddUrd) and 2',3'-dideoxy-3'-fluoro-5-hydroxymethyluridine (3'-F5HmddUrd), were synthesized. The reaction of 3'-azido-2',3'-dideoxyuridine (3'-AzddUrd) and 2',3'-dideoxy-3'-fluorouridine (3'-FddUrd) with formaldehyde, under strongly alkaline conditions and at elevated temperature, proceeded after 4 days to completion to afford the corresponding 5-hydroxymethyl derivatives 3'-Az5HmddUrd and 3'-F5HmddUrd in good yield. These compounds were also prepared by oxidation of AZT and FLT with the use of K2S2O8. 1H NMR analyses were subjected to the series of 3', 4 and 5-substituted pyrimidine 2'-deoxy- and 2',3'-dideoxynucleosides involving 3'-Az5HmddUrd and 3'-F5HmddUrd. Analysis of the sugar furanose ring puckering demonstrated that all 3'-fluorine derivatives exhibited strong domination of the S conformation (approximately 100%) while 3'-substitution by electron-donating groups, such as NH2, increased population of the N conformation. Experimentally observed substituent effect on the furanose ring puckering equilibrium was reconstructed in the 100 ps molecular dynamic trajectories obtained for AZT, FLT, dThd, 2',3'-ddThd and 3'-amino-2',3'-ddThd. It may be concluded that anti-HIV activity is linked to a direct interaction of the 3'-substituent with reverse transcriptase (RT) binding site. Anti-HIV activities of 3'-Az5HmddUrd and 3'-F5HmddUrd are lower than activity of AZT and FLT; however, 3'-Az5HmddUrd and 3'-F5HmddUrd are less toxic than AZT and FLT.
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PMID:Synthesis, solution conformation and anti-HIV activity of novel 3'-substituted-2',3'-dideoxy-5-hydroxymethyluridines and their 4,5-substituted analogues. 1452 29


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