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)

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

9-beta-D-Arabinofuranosyladenine (ara-A) is an inhibitor of DNA replication with antitumor and antiviral activity. The molecular basis for this inhibitory effect has remained unclear. The present work has examined the effects of 9-beta-D-arabinofuranosyladenine-triphosphate on DNA polymerase-beta. These studies were performed on different M13 phage DNA templates. The findings demonstrate that 9-beta-D-arabinofuranosyladenine is incorporated into the elongating DNA strand by DNA polymerase-beta. The findings also demonstrate that the incorporated 9-beta-D-arabinofuranosyladenine residue acts as a relative chain terminator. Furthermore, the relative chain-terminating effects of this agent are sequence specific and reversed by competition with deoxyadenosine-triphosphate for incorporation into the DNA strand. These findings are in concert with hydrogen bonding differences of the incorporated arabinosyl moiety which alters reactivity of the chain terminus and thereby inhibits elongation. These findings are also in agreement with recent studies of 1-beta-D-arabinofuranosylcytosine and provide insights into the sequence specific effects of these agents.
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PMID:Sequence-specific inhibition of DNA strand elongation by incorporation of 9-beta-D-arabinofuranosyladenine. 246 40

To investigate the molecular basis of the regulatory mechanisms responsible for the orderly replication of the mammalian genome, we have developed an experimental system by which the replication order of various genes can be defined with relative ease and precision. Exponentially growing CHO-K1 cells were separated into populations representing various stages of the cell cycle by centrifugal elutriation and analyzed for cell cycle status flow cytometry. The replication of specific genes in each elutriated fraction was measured by labeling with 5-mercuri-dCTP and [3H]dTPP under conditions of optimal DNA synthesis after cell permeabilization with lysolecithin. Newly synthesized mercurated DNA from each elutriated fraction was purified by affinity chromatography on thiol-agarose and replicated with the large fragment of Escherichia coli DNA polymerase I by using [alpha-32P]dATP and random primers. The 32P-labeled DNA representative of various stages of the cell cycle was then hybridized with dot blots of plasmid DNA containing specific cloned genes. From these results, it was possible to deduce the nuclear DNA content at the time each specific gene replicated during S phase (C value). The C values of 29 genes, which included single-copy genes, multifamily genes, oncogenes, and repetitive sequences, were determined and found to be distributed over the entire S phase. Of the 28 genes studied, 19 had been examined by others using in vivo labeling techniques, with results which agreed with the replication pattern observed in this study. The replication times of nine other genes are described here for the first time. Our method of analysis is sensitive enough to determine the replication time of single-copy genes. The replication times of various genes and their levels of expression in exponentially growing CHO cells were compared. Although there was a general correlation between transcriptional activity and replication in the first half of S phase, examination of specific genes revealed a number of exceptions. Approximately 25% of total poly(A) RNA was transcribed from the late-replicating DNA.
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PMID:Temporal order of gene replication in Chinese hamster ovary cells. 247 59

To investigate whether DNA replication in rat hepatoma cells is altered compared with that in normal rat liver, the main replicative enzyme, i.e. the DNA polymerase alpha complex, was partially purified from a slow-growing (TC5123) and a fast-growing (MH3924) Morris hepatoma cell strain as well as from normal rat liver. The purified DNA polymerase alpha complexes contained RNA primase. DNA polymerase alpha activities of these complexes were characterized with regard to both their molecular properties and their dNTP and DNA binding sites. The latter were probed with competitive inhibitors of dNTP binding, resulting in Ki values, and with DNA templates, yielding Km values. The sedimentation coefficients of native DNA polymerases alpha from Morris hepatoma cells were found to be lower than that of polymerase alpha from normal rat liver. Consequently, when following the procedure of Siegel and Monty for determination of molecular mass considerably smaller molecular masses were calculated for polymerases of hepatoma strains (TC5123, 127 kDa; MH3924, 138 kDa; rat liver, 168 kDa). Similar differences were found when the dNTP binding site was probed with inhibitors. Ki values obtained with butylphenyl-dGTP were higher for polymerases of the hepatoma strains than for that of normal rat liver. However, Ki values measured with aphidicolin and butylanilino-dATP were lower for DNA polymerase alpha from the fast-growing hepatoma cell strain than for that from normal rat liver, indicating a reduced affinity of the dNTP binding sites for dATP and dCTP. This reduced affinity could be responsible for lowered specificity of nucleotide selection in the base-pairing process which in turn may cause an enhanced error rate in DNA replication in malignant cells. Furthermore, when the DNA binding site was characterized by Michaelis-Menten constants using gapped DNA as a template, Km values were similar for all three DNA polymerases. In contrast, the Km value measured with single-stranded DNA as a template was found to be lower for DNA polymerase alpha from the fast-growing hepatoma MH3924 than for that from normal rat liver. Thus, the DNA-polymerizing complex from MH3924 combines both higher binding strength to single-stranded DNA templates and decreased nucleotide selection, properties which may enhance replication velocity and may lower fidelity.
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PMID:DNA polymerase alpha from normal rat liver is different than DNA polymerases alpha from Morris hepatoma strains. 250 1

The acyclic adenosine analogue (S)-9-(3-hydroxy-2-phosphonylmethoxypropyl)adenine [S]-HPMPA) is a potent and selective inhibitor of adenovirus (Ad) replication in cell culture. We studied the mechanism of inhibition using a reconstituted in vitro DNA replication system. The diphosphoryl derivative (S)-HPMPApp, but not (S)-HPMPA, inhibited the DNA replication of origin containing fragments strongly. The inhibitory effect was exerted at the level of elongation, while initiation was resistant to the drug. Remarkably, the elongation of short strands was only slightly impaired, while inhibition was maximal upon synthesis of long DNA fragments. (S)-HPMPApp appeared to be competitive with dATP, suggesting that the Ad DNA polymerase is the prime target for the drug. We purified the Ad DNA polymerase in complex to the precursor terminal protein to homogeneity from cells infected with overproducing recombinant vaccinia viruses. Employing gapped DNA or poly(dT).oligo(dA) templates, only a weak inhibition was observed. However, inhibition was strongly enhanced in the presence of the adenovirus DNA binding protein (DBP). We interpret this to mean that the increased processivity of the polymerization reaction in the presence of DBP leads to increased drug sensitivity.
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PMID:Mechanism of inhibition of adenovirus DNA replication by the acyclic nucleoside triphosphate analogue (S)-HPMPApp: influence of the adenovirus DNA binding protein. 258 48

Potential antiviral and antitumour nucleosides, 3'-fluoro-2', 3'-dideoxy-adenosine and -guanosine, have been synthesized by the chemical transglycosylation reaction using 5'-O-acetyl-3'-fluoro-2', 3'-dideoxy-thymidine and -uridine as donors of the carbohydrate fragment and persilylated 6-N-benzoyladenine and 2-N-palmitoylguanine as acceptors, respectively. 5'-Triphosphates of 3'-fluoro-2', 3'-dideoxy-thymidine, -cytidine, -adenosine, and -guanosine (dNTP(3'F] were synthesized and tested as terminators in cell-free system of DNA synthesis catalyzed by RNA-directed DNA polymerase (reverse transcriptase, RT) from the avian myeloblastosis virus (AMV) and E. coli DNA polymerase I (Klenow fragment). A method of estimating relative effectiveness of dNTP(3'F) incorporation into DNA growing chain in comparison with the natural substrates was developed. It is shown that, in case of AMV-RT, dATP(3'F), dCTP(3'F) incorporate 14 times less efficiently than dATP and dCTP respectively, and dTTP(3'F) 3 times less effectively than the corresponding natural substrates, whereas dGTP (3'F) is as efficient as dGTP. With E. coli DNA polymerase I (Klenow fragment) dATP (3'F) and dCTP(3'F) are ca. 100 times less efficient, and dTTP(3'F) and dGTP(3'F) are ca. 50 times less efficient than the respective natural substrates.
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PMID:[Synthesis of 2',3',-dideoxy-3'-fluoradenosine and -guanosine, their 5'-triphosphates and a study of 2',3'-dideoxy-3'-fluoronucleoside- 5'-triphosphates as substrates for DNA-polymerases]. 267 51

The tau and gamma subunits of the DNA polymerase III holoenzyme of Escherichia coli were each isolated in large quantities as oligomers from overproducing cells in which their genes (dnaZ and X) were under the control of a T7 phage promoter. The 52-kDa gamma subunit (encoded by the dnaZ sequence) contains three-forths of the N-terminal residues of the 71-kDa tau subunit (encoded by the dnaX sequence). Both gamma and tau share a binding site for ATP (or dATP). A DNA-dependent ATPase activity (Lee, S.H., and Walker, J.R. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 2713-2717) exhibited only by the tau subunit, presumably requires a DNA-binding site in the C-terminal domain lacking in the gamma subunit. Among ATPases dependent on single-stranded DNA, the tau activity is remarkable in the failure of homopolymers (e.g. poly(dA) or poly(dT)) to replace natural DNAs. The presumed need for certain secondary structures may reflect a feature of template binding in the crucial contribution that tau makes to the high processivity of polymerase III holoenzyme. Limited tryptic digestion of tau generates a fragment that resembles gamma in: (i) size, (ii) binding of ATP without ATPase activity, and (iii) a level of complementing holoenzyme activity in extracts of dnaZ-mutant cells that is higher than that of tau.
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PMID:ATP interactions of the tau and gamma subunits of DNA polymerase III holoenzyme of Escherichia coli. 268 Nov 83

The fluorescent nucleotide 2',3'-trinitrophenyl-ATP (TNP-ATP) binds at the triphosphate substrate binding site of the large (Klenow) fragment of DNA polymerase I (Pol I) as detected by direct binding studies measuring the increase in fluorescence of this ligand (n = 1.0, KD = 0.07 microM). The enzyme-TNP-ATP complex binds Mg2+ and Mn2+ tightly (KD = 0.05 microM) as measured by an increase in fluorescence on titrating with these metals. The substrate dGTP competitively displaces TNP-ATP from the enzyme (KD = 5.7 microM) de-enhancing the fluorescence. The polymerase reaction is half-maximally inhibited by 0.8 microM TNP-ATP in the presence of dATP (10 microM) as substrate. A region of the amino acid sequence of Pol I (peptide I) consisting of residues 728-777 has been synthesized and found to contain significant secondary structure by CD both in water and 50% methanol/water. In water at 3 degrees C, peptide I binds the substrate analog TNP-ATP (KD = 0.03 microM) with a stoichiometry of 0.2. In 50% methanol at 3 degrees C, peptide I binds TNP-ATP with a higher stoichiometry than in water, consistent with a 1:1 complex, but biphasically (16% of the peptide, KD = 0.09 microM; 84% of the peptide, KD = 5.0 microM), and competitively binds the Pol I substrates dATP, TTP, and dGTP (KD = 230-570 microM). Evidence from size exclusion high performance liquid chromatography suggests that these two forms of the peptide are monomer and dimer, respectively. Significantly, the peptide I-TNP-ATP complex binds duplex DNA, tightly (KD = 0.1-0.5 microM) and stoichiometrically, and single stranded DNA more weakly. The peptide I-duplex DNA complex binds both TNP-ATP (KD = 0.5-1.5 microM) and Pol I substrates (KD = 350-2100 microM) stoichiometrically. In a control experiment, a second peptide, peptide II, based on residues 840-888 of the Pol I sequence, retains secondary structure, as detected by CD, but displays no binding of TNP-ATP. The ability of peptide I, which represents only 8% of the large fragment of Pol I, to bind both substrates and duplex DNA indicates that residues 728-777 constitute a major portion of the substrate binding site of this enzyme.
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PMID:Substrate and DNA binding to a 50-residue peptide fragment of DNA polymerase I. Comparison with the enzyme. 268 60

We developed a system for rapid, manual and automated sequence analysis by utilizing and modifying methods used in conjunction with the polymerase chain reaction (PCR). We are using these techniques to detect single base mutations in the dihydrofolate reductase (DHFR) gene giving rise to methotrexate (MTX) resistance of tumor cells obtained from patients with malignancies. Amplifying in vitro both genomic DNA and transcripts of the human DHFR we are able to reproducibly generate single-stranded templates. Utilizing [alpha-35S]dATP and both the universal and reverse sequencing primers we obtain sequence information from either strand. The methods described have been successfully used for automated sequencing with the Applied Biosystems Model 370A Sequencer using both modified T7 DNA polymerase and Taq I. DNA polymerase for dideoxy-termination sequencing. The use of this methodology to detect a single base change in a human colon carcinoma cell line, HCT-8, is illustrated.
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PMID:Sequence analysis of a human gene responsible for drug resistance: a rapid method for manual and automated direct sequencing of products generated by the polymerase chain reaction. 269 61

A photoaffinity analogue of dATP, 8-azido-2'-deoxyadenosine 5'-triphosphate (8-azido-dATP), was used to probe the nucleotide binding site of the non-template-directed DNA polymerase terminal deoxynucleotidyl transferase (EC 2.7.7.31). The Mg2+ form of 8-azido-dATP was shown to be an efficient enzyme substrate with a Km of 53 microM. Loss of enzyme activity occurred during UV photolysis only in the presence of 8-azido-dATP. At saturation (120 microM 8-azido-dATP), 54% of the protein molecules were modified as determined by inhibition of enzyme activity. Kinetic analysis of enzyme inhibition induced by photoincorporation of 8-azido-dATP indicated an apparent Kd of approximately 38 microM. Addition of 2 mM dATP to 120 microM 8-azido-dATP resulted in greater than 90% protection from photoinduced loss of enzyme activity. In contrast, no protection was observed with the addition of 2 mM dAMP. Enzyme inactivation was directly correlated with incorporation of radiolabeled 8-azido-dATP into the protein and UV-induced destruction of the azido group. Photoincorporation of 8-azido-dATP into terminal transferase was reduced by all purine and pyrimidine deoxynucleoside triphosphates of which dGTP was the most effective. The alpha and beta polypeptides of calf terminal transferase were specifically photolabeled by [gamma-32P]-8-azido-dATP, and both polypeptides were equally protected by all four deoxynucleoside triphosphates. This suggests that the nucleotide binding domain involves components from both polypeptides.
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PMID:Photoaffinity labeling of terminal deoxynucleotidyl transferase. 1. Active site directed interactions with 8-azido-2'-deoxyadenosine 5'-triphosphate. 271 38


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