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)

Rubromycins, a class of quinone antibacterials, were discovered to selectively inhibit human immunodeficiency virus-1 (HIV-1) RNA-directed DNA polymerase (reverse transcriptase) (RT) activity more potently than cellular DNA polymerase alpha. beta- and gamma-rubromycin each inhibited equipotently HIV-1 RT and avian myeloblastosis virus RT, in a concentration-dependent manner, and were significantly weaker as inhibitors of calf thymus DNA polymerase alpha. These agents inhibited HIV-1 RT reversibly, were competitive with respect to template.primer, and were noncompetitive with respect to TTP. Dixon analyses yielded HIV RT Ki values of 0.27 +/- 0.014 and 0.13 +/- 0.012 microM for beta- and gamma-rubromycin, respectively. Similarly, using DNA polymerase alpha, the Ki values were 25.1 +/- 4.3 and 3.9 +/- 0.6 microM for beta- and gamma-rubromycin, respectively. Because these agents were toxic to noninfected human T lymphoid cells using concentrations at or above 6 microM, HIV-1 infectivity studies were carried out at 0.8-6 microM. At these concentrations, which are below the range expected to provide protection, no significant antiviral activity was observed. Although beta- and gamma-rubromycins did not possess sufficient HIV RT inhibitory potency or selectivity versus mammalian DNA polymerase to demonstrate antiviral activities, these studies support the hypothesis that specific molecules containing quinone functional groups can selectively inhibit viral polymerase activities over cellular polymerase activities. In addition, these studies suggest that rubromycins may be lead structures for the development of more potent and selective agents.
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PMID:Inhibition of human immunodeficiency virus-1 reverse transcriptase activity by rubromycins: competitive interaction at the template.primer site. 169 17

We have used an in vitro assay to study the induction of DNA synthesis by cytoplasmic extracts from the actively growing cell line Molt 4 in nuclei isolated from quiescent human lymphocytes. The TTP incorporation which takes place in these nuclei has been shown to be inhibitable by serine protease inhibitors, particularly aprotinin. This DNA synthesis has also been proposed to reflect the initiation of true DNA replication; however, we find evidence that much, if not most, of this incorporation is due to nonreplicative synthesis initiated on primer templates formed by calcium-dependent activation of the nuclear chromatin substrate. The principal DNA polymerase supplied by the Molt 4 extract appears to be polymerase alpha and the results show that the activated chromatin is a substrate for purified bacterial DNA polymerases. DNA synthesis is significantly enhanced by preincubation at 37 degrees C in the presence of calcium, and the almost complete inhibition of DNA synthesis induced by extracts or bacterial polymerases in the presence of T4 ligase suggests that this chromatin activation involves calcium-dependent endonucleases. Nevertheless, DNA synthesis in the isolated nuclei, with both Molt 4 extracts and bacterial polymerases, is substantially inhibited by addition of serine protease inhibitors, with aprotinin the most potent of those tested on a molar basis. Thus, the results suggest that specific proteolytic activity is required before nicked or damaged nuclear DNA can serve as an acceptable substrate for DNA polymerase activity.
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PMID:DNA synthesis in isolated resting nuclei: evidence for protease-dependent nonreplicative nucleotide incorporation. 169 99

2-Chloro-9-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-adenine (Cl-F-ara-A) has activity against the P388 tumor in mice on several different schedules. Biochemical studies with a chronic myelogenous leukemia cell line (K562) grown in cell culture have been done in order to better understand its mechanism of action. Cl-F-ara-A was a potent inhibitor of K562 cell growth. Only 5 nM inhibited K562 cell growth by 50% after 72 h of continuous incubation. The 5'-triphosphate of Cl-F-ara-A was detected by strong anion exchange chromatography of the acid-soluble extract of K562 cells incubated with Cl-F-ara-A. Competition studies with natural nucleosides suggested that deoxycytidine kinase was the enzyme responsible for the metabolism to the monophosphate. Incubation of K562 cells for 4 h with 50 nM Cl-F-ara-A inhibited the incorporation of [3H]thymidine into the DNA by 50%. Incubation with 0.1, 1, or 10 microM Cl-F-ara-A for 4 h depressed dATP, dCTP, and dGTP pools but did not affect TTP pools. Similar inhibition of deoxyribonucleoside triphosphate pools was seen after incubation with 2-chloro-2'-deoxyadenosine. Both Cl-F-ara-ATP and Cl-dATP potently inhibited the reduction of ADP to dADP in crude extracts of K562 cells (concentration producing 50% inhibition, 65 nM). The effect of Cl-F-ara-ATP on human DNA polymerases alpha, beta, and gamma isolated from K562 cells grown in culture was determined and compared with those of Cl-dATP and 9-beta-D-arabinofuranosyl-2-fluoroadenine triphosphate (F-ara-ATP). Cl-F-ara-ATP was a potent inhibitor of DNA polymerase alpha. Inhibition of DNA polymerase alpha was competitive with respect to dATP (Ki of 1 microM). The three analogue triphosphates were incorporated into the DNA by DNA polymerase alpha as efficiently as dATP. The incorporation of Cl-F-ara-AMP inhibited the further elongation of the DNA chain, similarly to that seen after the incorporation of F-ara-AMP. Extension of the DNA chain after the incorporation of Cl-dAMP was not inhibited as much as it was with either Cl-F-ara-AMP or F-ara-AMP. Cl-F-ara-ATP was not a potent inhibitor of DNA polymerase beta, DNA polymerase gamma, or DNA primase.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Effects of 2-chloro-9-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)adenine on K562 cellular metabolism and the inhibition of human ribonucleotide reductase and DNA polymerases by its 5'-triphosphate. 170 52

3'-Mercapto-3'-deoxy-TTP was synthesized and tested as DNA chain terminator nucleotide for calf thymus alpha DNA polymerase, E. coli DNA polymerase I (Klenow fragment), terminal deoxyribonucleotidyltransferase (Bollum enzyme) and reverse transcriptase from AMV- and HIV-I-viruses. It was shown that the compound terminates DNA chain elongation by reverse transcriptases selectively and irreversibly. Other tested DNA polymerases do not use this nucleotide analogue as a substrate. 3'-Mercapto-3'-deoxythymidine was tested on lymphoblastoid T-cell line MT-4 with HIV-viruses and shown to suppress viruses as efficiently as 3'-azido-3'-deoxythymidine.
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PMID:[3'-Mercapto-3'-deoxythymidine-5'-triphosphate as a terminator of DNA synthesis catalyzed by RNA-dependent DNA-polymerases]. 171 4

Psychotrine dihydrogen oxalate and O-methylpsychotrine sulfate heptahydrate (MP), the salts of isoquinoline alkaloids from ipecac, were found to be potent inhibitors of the DNA polymerase activity of human immunodeficiency virus-1 reverse transcriptase (HIV-1 RT). We currently report the results of additional studies designed to characterize the mechanism of inhibition facilitated by MP. The inhibition was noncompetitive with respect to TTP and uncompetitive with respect to poly(rA) and oligo(dT)12-18 (4:1) at low template-primer concentrations but competitive at high concentrations (greater than 200 microM). Identical non-Michaelis-type kinetics were observed when activated DNA was used as the template. The biphasic nature of the double-reciprocal plots and Hill coefficients of less than 1 indicate that MP functions as an allosteric inhibitor of the enzyme which appears to possess multiple active sites that interact in a cooperative (negative) fashion in the presence of the inhibitor. MP was selective for the recombinant HIV-1 RT (p66) utilizing poly(rA) and oligo(dT)12-18 (4:1) as template-primer. Greater inhibition was observed with this template primer as compared with other natural and synthetic template-primers tested. MP had significantly less effect on avian myeloblastosis virus RT as well as mammalian or bacterial DNA and RNA polymerases. Other members of the ipecac class of alkaloids, e.g. emetine hydrochloride, were inactive against all of these enzymes, including HIV-1 RT. Conversely, MP did not inhibit in vitro protein synthesis, a property manifested by all the other ipecac alkaloids tested. Studies conducted with structural analogs revealed that the imine functionality at positions 1' and 2' of MP is the key structural requirement for HIV-1 RT inhibitory activity. Therefore, MP appears to possess unique structural properties that enable interaction with HIV-1 RT in a manner that can be differentiated from other polymerases. Use of these alkaloids for the definition of this viral enzyme-specific topology may lead to the development of therapeutically useful chemotherapeutic agents.
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PMID:Psychotrine and its O-methyl ether are selective inhibitors of human immunodeficiency virus-1 reverse transcriptase. 172 Oct 50

A DNA primase activity was isolated from pea chloroplasts and examined for its role in replication. The DNA primase activity was separated from the majority of the chloroplast RNA polymerase activity by linear salt gradient elution from a DEAE-cellulose column, and the two enzyme activities were separately purified through heparin-Sepharose columns. The primase activity was not inhibited by tagetitoxin, a specific inhibitor of chloroplast RNA polymerase, or by polyclonal antibodies prepared against purified pea chloroplast RNA polymerase, while the RNA polymerase activity was inhibited completely by either tagetitoxin or the polyclonal antibodies. The DNA primase activity was capable of priming DNA replication on single-stranded templates including poly(dT), poly(dC), M13mp19, and M13mp19 + 2.1, which contains the AT-rich pea chloroplast origin of replication. The RNA polymerase fraction was incapable of supporting incorporation of 3H-TTP in in vitro replication reactions using any of these single-stranded DNA templates. Glycerol gradient analysis indicated that the pea chloroplast DNA primase (115-120 kDa) separated from the pea chloroplast DNA polymerase (90 kDa), but is much smaller than chloroplast RNA polymerase. Because of these differences in size, template specificity, sensitivity to inhibitors, and elution characteristics, it is clear that the pea chloroplast DNA primase is an distinct enzyme form RNA polymerase. In vitro replication activity using the DNA primase fraction required all four rNTPs for optimum activity. The chloroplast DNA primase was capable of priming DNA replication activity on any single-stranded M13 template, but shows a strong preference for M13mp19 + 2.1. Primers synthesized using M13mp19 + 2.1 are resistant to DNase I, and range in size from 4 to about 60 nucleotides.
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PMID:Pea chloroplast DNA primase: characterization and role in initiation of replication. 186 57

Oligonucleotides were annealed to complementary sequences in fixed human metaphase chromosomes and extended with DNA polymerase. The newly synthesized fragments were labeled by incorporating bio-11-dUTP instead of TTP, and the sites of synthesis were detected by immunocytochemistry, using fluorochromes as the reporter molecules. We have obtained clear localization with oligonucleotides from alphoid (centromeric sequences), simple sequence (satellite) DNAs, a variety of Alu-dispersed repeated sequences, and oligonucleotides derived from the Tetrahymena and Trypanosoma telomere-specific sequences. The simple sequence and alphoid oligonucleotides gave results at least comparable to those obtained using the whole molecule as a probe for in situ hybridization, whereas the Alu oligonucleotides produced a diversity of results which depended on the absolute length and location of the oligonucleotide within the Alu sequence. The telomere-specific oligomers also produced a variety of results. The G-rich Trypanosoma oligomer and its complementary C-rich sequence produced strong telomeric signals and some interstitial signals on mouse chromosomes, but only weak telomeric signals on human chromosomes. The G-rich Tetrahymena oligomer produced detectable telomeric signals on human chromosomes. The technique appears to be a valuable extension of present tools for mapping and examining the organization of DNA sequences within chromosomes.
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PMID:Oligonucleotide-primed in situ DNA synthesis (PRINS): a method for chromosome mapping, banding, and investigation of sequence organization. 191 15

We have constructed a plasmid that, when introduced into Escherichia coli, induces the synthesis of large quantities of a protein with an apparent molecular mass of 66 kDa that differs from human immunodeficiency virus (HIV) RNA-dependent DNA polymerase (deoxynucleoside-triphosphate:DNA deoxynucleotidyltransferase or reverse transcriptase, EC 2.7.7.49) only in that it has two additional amino-terminal amino acids. This protein is soluble in E. coli extracts, is active in reverse transcriptase assays, and shows inhibition profiles with dideoxy-TTP and dideoxy-GTP that are indistinguishable from the viral enzyme. The deletion of 23 amino-terminal or carboxyl-terminal amino acids or the insertion of 5 amino acids at position 143 substantially decreases the polymerizing activity of the HIV reverse transcriptase made in E. coli. The properties of a 51-kDa reverse transcriptase-related protein made in E. coli suggests that the p51 found in the virion probably does not have substantial polymerizing activity. The full-length HIV reverse transcriptase and the various mutant proteins produced in E. coli should be quite useful for structural and biochemical analyses as well as for the production of antibodies.
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PMID:Expression of soluble, enzymatically active, human immunodeficiency virus reverse transcriptase in Escherichia coli and analysis of mutants. 244 94

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

Several analogues of 2',3'-dideoxythymidine 5'-triphosphate [i.e., 3'-azido-2', 3'-dideoxythymidine 5'-triphosphate(Azdd TTP), 2',3'-didehydro-2',3'-dideoxythymidine 5'-triphosphate (ddeTTP), alpha, beta-methylene 3'-azido-2',3'-dideoxythymidine 5'-diphosphate, alpha, beta-methylene 3'-azido-2',3'-dideoxythymidine 5'-triphosphate, and beta, gamma-methylene 3'-azido-2',3'-dideoxythymidine 5'-triphosphate] and 2',3'-didehydro-2',3'-dideoxycytidine 5'-triphosphate (ddeCTP) have been evaluated for their inhibitory effects on murine retroviral reverse transcriptase and various other DNA polymerases, including DNA polymerases alpha, beta, and gamma, terminal deoxynucleotidyl transferase, and DNA polymerase I. None of the compounds inhibited the activity of DNA polymerase alpha under the reaction conditions employed. When Mg2+ was replaced by Mn2+, however, DNA polymerase alpha was strongly inhibited only by ddeTTP. DNA polymerase beta activity was inhibited only by ddeTTP and ddeCTP. All the compounds, except for ddeCTP, inhibited DNA polymerase gamma activity, ddeTTP being a particularly strong inhibitor of gamma-polymerase (Ki = 3.5 nM). Terminal deoxynucleotidyl transferase was only slightly inhibited by any of the compounds. AzddTTP was a potent inhibitor of reverse transcriptase (Ki = 42 nM), but it also inhibited the activities of DNA polymerase gamma and DNA polymerase I.
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PMID:Differential inhibitory effects of several pyrimidine 2',3'-dideoxynucleoside 5'-triphosphates on the activities of reverse transcriptase and various cellular DNA polymerases. 247 Oct 54


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