Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0021051 (immunodeficiency)
 71,517 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Nondenaturing gel electrophoresis was used to study the nucleotide substrate-induced conformational change in reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1). Dead-end complex was formed between HIV-1 RT, dideoxynucleotide chain-terminated primer, and DNA template in the presence of deoxynucleotide triphosphate (dNTP) complementary to the next position on the template. Complexes which form in the absence of the next complementary dNTP were disrupted by adding excess poly(rA)/oligo(dT) or heparin just prior to electrophoresis. Dead-end complex formation by noncomplementary dNTP's or ribonucleotides was at least 2000-fold less efficient than with the complementary nucleotide. When dA was the next nucleotide on the template, analogues of dTTP supported dead-end complex formation with increased apparent Kd (dTTP < dideoxy-TTP approximately alpha-thio-dTTP < dUTP < 3'-azidothymidine triphosphate). A similar relationship was observed for dGTP analogues across from dC on the template (dGTP < dideoxy-GTP < alpha-thio-dGTP << dITP < dideoxy-ITP). The optimal length of the primer/template duplex region for dead-end complex formation was between 20 and 32 base pairs. Primer-template with a mismatched primer terminus did not support dead-end complex formation, and primer terminated with 3'-azidothymidine formed dead-end complex with 25-fold elevated apparent Kd. By contrast, dead-end complex formation on primer terminated with dideoxy-IMP base paired with dC on the template was more efficient than on primer terminated with dideoxy-GMP. Implications for the mechanisms of discrimination between nucleotide analogues by HIV-1 RT are discussed.
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PMID:Nucleotide-induced stable complex formation by HIV-1 reverse transcriptase. 915 15

The effects of deoxynucleoside triphosphate (dNTP) imbalances on the fidelity of human immunodeficiency virus type 1 (HIV-1) replication were investigated. Using detergent permeabilized virions and biased dNTP concentrations different types of hypermutants were readily produced. However, the mutant spectrum was different from naturally occurring hypermutants demonstrating that the host cell may restrict variation. Using a genetic screen based on the blue/white beta-galactosidase complementation assay, G --> A hypermutants were recovered from HIV-infected thymidine treated U937 cells. Furthermore, hypermutants were recovered from 1 to 2% of resting or activated peripheral blood mononuclear cells indicating that small proportions of primary cells had distorted intracellular [dTTP] and [dCTP]. Such imbalances may underlie a proportion of somatic and germline point mutations and shape to some extent the evolution of mammalian and viral genomes.
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PMID:HIV genetic variation is directed and restricted by DNA precursor availability. 923 17

The carcinogen ethylene dibromide (EDB) has been shown to cause glutathione (GSH)-dependent base-substitution mutations, especially GC to AT transitions, in a variety of bacterial and eukaryotic systems. The known DNA adducts S-[2-(N7-guanyl)ethyl]GSH, S-[2-(N2-guanyl)ethyl]GSH, and S-[2-(O6-guanyl)ethyl]GSH were individually placed at a site in a single oligonucleotide. Polymerase extension studies were carried out using Escherichia coli polymerase I exo- (Klenow fragment, Kf-) and polymerase II exo- (pol II-), bacteriophage T7 polymerase exo-, and human immunodeficiency virus-1 reverse transcriptase in order to characterize misincorporation events. Even though extension was not as efficient as with the nonadducted template, some fully extended primers were observed with the template containing S-[2-(N7-guanyl)ethyl]GSH using all of these polymerases. dCTP was the most preferred nucleotide incorporated opposite S-[2-(N7-guanyl)ethyl]GSH by most of polymerases examined; however, dTTP incorporation was observed opposite S-[2-(N7-guanyl)ethyl]GSH with pol II-. Both S-[2-(N2-guanyl)ethyl]GSH and S-[2-(O6-guanyl)ethyl]GSH strongly blocked replication by all polymerases. Only dATP and dGTP were incorporated opposite S-[2-(N2-guanyl)ethyl]GSH by both Kf- and pol II-. S-[2-(O6-Guanyl)ethyl]GSH was shown to strongly code for dATP incorporation by Kf-. With pol II-, dTTP was incorporated opposite S-[2-(O6-guanyl)ethyl]GSH. In conclusion, all three GSH-guanyl adducts derived from the carcinogen EDB blocked the polymerases and were capable of miscoding.
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PMID:Polymerase blockage and misincorporation of dNTPs opposite the ethylene dibromide-derived DNA adducts S-[2-(N7-guanyl)ethyl]glutathione, S-[2-(N2-guanyl)ethyl]glutathione, and S-[2-(O6-guanyl)ethyl]glutathione. 954 1

A sulfated glycoglycerolipid, 1-O-(6'-sulfo-alpha-D-glucopyranosyl)-2,3-di-O-phytanyl- sn-glycerol (KN-208), a derivative of the polar lipid isolated from an archaebacterium, strongly inhibited DNA polymerase (pol) alpha and pol beta in vitro among 5 eukaryotic DNA polymerases (alpha, beta, gamma, delta, and epsilon). It also inhibited Escherichia coli DNA polymerase I Klenow fragment (E. coli pol I) and human immunodeficiency virus reverse transcriptase (HIV RT). The mode of inhibition of these polymerases was competitive with the DNA template primer and was non-competitive with the substrate dTTP. KN-208 inhibited pol beta most strongly, with a Ki value of 0.05 microM, 10-fold lower than that for pol alpha (0.5 microM) and 60- or 140-fold lower than that for HIV RT (3 microM) or for E. coli pol I (7 microM), respectively. The loss of sulfate on the 6'-position of glucopyranoside of this compound completely abrogated inhibition. However, the hydrophilic part of KN-208, glucose 6-sulfate alone, showed no inhibition. Other sulfated compounds containing different hydrophobic structures, such as dodecyl sulfate and cholesterol sulfate, exhibited a much weaker inhibition. Our results suggest that the whole molecular structure of KN-208 is required for inhibition. KN-208 was shown to be modestly cytotoxic for the human leukemic cell line K562. Interestingly, a subcytotoxic dose of KN-208 increased the sensitivity of the human leukemic cells to an alkylating agent, methyl methanesulfonate, while it did not potentiate the effects of ultraviolet light or of cisplatin.
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PMID:Sulfated glycoglycerolipid from archaebacterium inhibits eukaryotic DNA polymerase alpha, beta and retroviral reverse transcriptase and affects methyl methanesulfonate cytotoxicity. 959 Jan 27

Telomeric DNA consists of short, tandemly repeated sequences at the ends of chromosomes. Telomeric DNA in the ciliate Paramecium tetraurelia is synthesized by an error-prone telomerase with an RNA template specific for GGGGTT repeats. We have previously shown that misincorporation of TTP residues at the telomerase RNA templating nucleotide C52 accounts for the 30% GGGTTT repeats randomly distributed in wild-type telomeres. To more completely characterize variable repeat synthesis in P. tetraurelia, telomerase RNA genes mutated at C52 (A, U, and G) were expressed in vivo. De novo telomeric repeats from transformants indicate that the predominant TTP misincorporation error seen in the wild-type telomerase is dependent on the presence of a C residue at template position 52. Paradoxically, the effects of various other telomerase RNA template and alignment region mutations on de novo telomeres include significant changes in fidelity, as well as the synthesis of aberrant, 5-nucleotide telomeric repeats. The occurrence of deletion errors and the altered fidelity of mutated P. tetraurelia telomerase, in conjunction with misincorporation by the wild-type enzyme, suggest that the telomerase RNA template domain may be analogous to homopolymeric mutational hot spots that lead to similar errors by the human immunodeficiency virus proofreading-deficient reverse transcriptase.
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PMID:Expression of mutated Paramecium telomerase RNAs in vivo leads to templating errors that resemble those made by retroviral reverse transcriptase. 1008 55

(+)-Calanolide A (NSC 650886) has previously been reported to be a unique and specific nonnucleoside inhibitor of the reverse transcriptase (RT) of human immunodeficiency virus (HIV) type 1 (HIV-1) (M. J. Currens et al., J. Pharmacol. Exp. Ther., 279:645-651, 1996). Two isomers of calanolide A, (-)-calanolide B (NSC 661122; costatolide) and (-)-dihydrocalanolide B (NSC 661123; dihydrocostatolide), possess antiviral properties similar to those of calanolide A. Each of these three compounds possesses the phenotypic properties ascribed to the pharmacologic class of nonnucleoside RT inhibitors (NNRTIs). The calanolide analogs, however, exhibit 10-fold enhanced antiviral activity against drug-resistant viruses that bear the most prevalent NNRTI resistance that is engendered by amino acid change Y181C in the RT. Further enhancement of activity is observed with RTs that possess the Y181C change together with mutations that yield resistance to AZT. In addition, enzymatic inhibition assays have demonstrated that the compounds inhibit RT through a mechanism that affects both the K(m) for dTTP and the V(max), i.e., mixed-type inhibition. In fresh human cells, costatolide and dihydrocostatolide are highly effective inhibitors of low-passage clinical virus strains, including those representative of the various HIV-1 clade strains, syncytium-inducing and non-syncytium-inducing isolates, and T-tropic and monocyte-tropic isolates. Similar to calanolide A, decreased activities of the two isomers were observed against viruses and RTs with amino acid changes at residues L100, K103, T139, and Y188 in the RT, although costatolide exhibited a smaller loss of activity against many of these NNRTI-resistant isolates. Comparison of cross-resistance data obtained with a panel of NNRTI-resistant virus strains suggests that each of the three stereoisomers may interact differently with the RT, despite their high degree of structural similarity. Selection of viruses resistant to each of the three compounds in a variety of cell lines yielded viruses with T139I, L100I, Y188H, or L187F amino acid changes in the RT. Similarly, a variety of resistant virus strains with different amino acid changes were selected in cell culture when the calanolide analogs were used in combination with other active anti-HIV agents, including nucleoside and nonnucleoside RT and protease inhibitors. In assays with combinations of anti-HIV agents, costatolide exhibited synergy with these anti-HIV agents. The calanolide isomers represent a novel and distinct subgroup of the NNRTI family, and these data suggest that a compound of the calanolide A series, such as costatolide, should be evaluated further for therapeutic use in combination with other anti-HIV agents.
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PMID:Unique anti-human immunodeficiency virus activities of the nonnucleoside reverse transcriptase inhibitors calanolide A, costatolide, and dihydrocostatolide. 1042 99

Polycitone A, an aromatic alkaloid isolated from the ascidian Polycitor sp. exhibits potent inhibitory capacity of both RNA- and DNA-directed DNA polymerases. The drug inhibits retroviral reverse transcriptase (RT) [i.e. of human immunodeficiency virus type 1 (HIV), murine leukaemia virus (MLV) and mouse mammary tumour virus (MMTV)] as efficiently as cellular DNA polymerases (i.e. of both DNA polymerases alpha and beta and Escherichia coli DNA polymerase I). The mode and mechanism of inhibition of the DNA-polymerase activity associated with HIV-1 RT by polycitone A have been studied. The results suggest that the inhibitory capacity of the DNA polymerase activity is independent of the template-primer used. The RNase H function, on the other hand, is hardly affected by this inhibitor. Polycitone A has been shown to interfere with DNA primer extension as well as with the formation of the RT-DNA complex. Steady-state kinetic studies demonstrate that this inhibitor can be considered as an allosteric inhibitor of HIV-1 RT. The target site on the enzyme may be also spatially related to the substrate binding site, since this inhibitor behaves competitively with respect to dTTP with poly(rA).oligo(dT) as template primer. Chemical transformations of the five phenol groups of polycitone A by methoxy groups have a determinant effect on the inhibitory potency. Thus, the pentamethoxy derivative which is devoid of all hydroxy moieties, loses significantly, by 40-fold, the ability to inhibit the DNA polymerase function. Furthermore, this analogue lacks the ability to inhibit DNA primer extension as well as the formation of the RT-DNA complex. Indeed, inhibition of the first step in DNA polymerization, the formation of the RT-DNA complex, and hence, of the overall process, could serve as a model for a universal inhibitor of the superfamily of DNA polymerases.
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PMID:Polycitone A, a novel and potent general inhibitor of retroviral reverse transcriptases and cellular DNA polymerases. 1054 37

Among the clinically used nucleoside analogue inhibitors that target human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT), there is little detailed mechanistic information on the interactions of 2',3'-didehydro-2', 3'-dideoxythymidine-5'-triphosphate (d4TTP) with the enzyme. primer-template complex and how these interactions compare with those of the natural substrate, dTTP. Using a pre-steady-state kinetic analysis, we found that d4TTP was incorporated by HIV-1 RT just as efficiently as dTTP during both DNA- and RNA-dependent DNA synthesis. To our knowledge, these results represent the first observation of a 3'-modified nucleoside triphosphate analogue that has an incorporation efficiency comparable to that observed for the natural substrate during DNA synthesis by HIV-1 RT. This information provides a mechanistic basis for understanding the inhibition of HIV-1 RT by d4TTP as well as insight into the clinically observed lack of d4T resistance mutations in HIV-1 RT isolated from AIDS patients.
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PMID:Mechanism of inhibition of the human immunodeficiency virus type 1 reverse transcriptase by d4TTP: an equivalent incorporation efficiency relative to the natural substrate dTTP. 1060 55

Adenosine deaminase deficiency is an inborn error resulting in immunodeficiency. The pathogenesis of the lymphopenia is not fully understood. Intracellular increases in dATP in the absence of deamination retard DNA repair in human resting lymphocytes and results in the slow accumulation of DNA strand breaks. We focused on the relationship between DNA damage and DNA precursor pools in cultures of deoxycoformycin-treated, ADA-inhibited resting lymphocytes. The addition of 10 microM deoxyadenosine led to a substantial number of DNA strand breaks within 12 h, breaks equivalent to those which occur with about 190 rad irradiation. Addition of any of the other deoxynucleosides used partially prevented this dAdo-induced DNA damage and promoted DNA repair. However, the preventive effects did not correlate inversely with intracellular dATP levels. Resting lymphocytes have very small dNTP pools. Treatment with dAdo slightly reduced dTTP and dCTP. Three kinds of deoxynucleosides, other than dAdo, restored or raised the corresponding dNTP level but the pool imbalance was only minimally corrected. Regarding the toxic effects of dAdo in ADA deficiency, not only dATP levels but also dNTP pool balance has a crucial role in the pathogenesis. Pool sizes of dTTP, dCTP, and possibly dGTP must be maintained at normal levels, if dAdo-induced DNA damage is to be avoided.
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PMID:Protection by various deoxynucleosides against deoxyadenosine-induced DNA damage in adenosine deaminase-inactivated lymphocytes. 1060 74

Initiation of human immunodeficiency virus-1 reverse transcription requires formation of a complex containing the viral RNA, primer tRNA(3)(Lys), and reverse transcriptase. Initiation, corresponding to addition of the first six nucleotides to tRNA(3)(Lys), is distinguished from elongation by its high specificity and low efficiency (processivity). Here, we compared the inhibition of initiation and elongation of reverse transcription by 3'-azido-3'-deoxythymidine 5'-triphosphate (AZTTP), the active form of 3'-azido-3'-deoxythymidine. We report the first detailed study of nucleotide binding, discrimination, and pyrophosphorolysis by the authentic initiation complex. We showed that the initiation and elongation complexes bound AZTTP and dTTP with the same affinity, while the polymerization rates were reduced by 148-160-fold during initiation. The pyrophosphorolysis rate of dTTP was reduced by the same extent, indicating that the polymerization equilibrium is the same in the two phases. The efficient unblocking of the 3'-azido-3'-deoxythymidine 5'-monophosphate (AZTMP)-terminated primer by pyrophosphorolysis significantly relieved inhibition of DNA synthesis during elongation in the presence of physiological pyrophosphate concentrations. Remarkably, although pyrophosphorolysis of dTMP and AZTMP were equally efficient during elongation, reverse transcriptase was almost totally unable to unblock the AZTMP-terminated primer during initiation. As a result, inhibition of reverse transcription by AZTTP was more efficient during initiation than elongation of reverse transcription, despite a reduced selectivity of incorporation.
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PMID:Inhibition of the initiation of HIV-1 reverse transcription by 3'-azido-3'-deoxythymidine. Comparison with elongation. 1086 29


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