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)

We address the question of whether substituting 2-aminopurine (APur) in place of adenine (Ade) in DNA can increase the frequency of base mispairing with cytosine. Using DNA polymerase alpha to measure the rates of inserting deoxycytidine and thymidine nucleotides in direct competition with each other for APur or Ade sites on synthetic copolymer DNA templates, we observe that the ratio of dCMP to dTMP insertion is increased by a factor of at least 230 when APur replaces Ade on a poly(dA) template and by a factor of 35 when APur replaces Ade on a poly(dC,dA) template. These data support the idea that APur.C base mispairs are directly involved in APur induction of A.T leads to G.C transition mutations. The observed misinsertion frequency of cytosine substituting for thymine opposite template APur sites is about 5%. This value is in excellent agreement with earlier predictions and measurements for APur.C heteroduplex-heterozygote frequencies in T4 bacteriophage in vivo.
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PMID:On the molecular basis of transition mutations: frequencies of forming 2-aminopurine.cytosine and adenine.cytosine base mispairs in vitro. 694 7

The 80,000-dalton form of the adenovirus (Ad) terminal protein (pTP) has been purified from Ad-infected HeLa cells. pTP was assayed by its ability to form a covalent complex with dCMP. The protein copurified with an activity that is essential for in vitro Ad DNA replication (Ad protein activity) as well as with a DNA polymerase activity that was distinguished from those of HeLa cell DNA polymerases alpha, beta, and gamma. The Ad protein-associated DNA polymerase activity was detected with activated DNA but not with poly(rA).oligo(dT) as template and was insensitive to aphidicolin and sensitive to N-ethylmaleimide. The Ad protein, DNA polymerase, and pTP-dCMP complex-forming activities sedimented in a glycerol gradient as a single peak with an apparent molecular size of 180,000 daltons. NaDodSO4/polyacrylamide gel analysis of the glycerol gradient fraction showed major bands of 80,000 and 140,000 daltons. The 80,000-dalton band was identified as pTP by comparison of its tryptic peptide map with that of the 55,000-dalton form of the terminal protein, which was purified from Ad virions.
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PMID:Adenovirus DNA replication in vitro: purification of the terminal protein in a functional form. 694 51

A complex containing the 80,000-dalton precursor to the adenovirus (Ad)-encoded terminal protein (pTP) and a 140,000-dalton protein is required for Ad DNA replication in vitro. This complex has been separated into subunits by glycerol gradient centrifugation in the presence of urea. The isolated 140,000-dalton subunit contains a DNA polymerase activity which can be differentiated from all host DNA polymerases. No enzyme activity was detected with the isolated pTP. The requirements for reactions involved in the initiation of Ad DNA replication were determined by using the isolated subunits. The covalent addition of dCMP, the first nucleotide in the DNA chain, to the pTP, which serves as the primer for replication, required the DNA polymerase subunit as well as the pTP. Synthesis of viral DNA in vitro also required both subunits. The properties of the DNA polymerase suggest that it may be a viral gene product.
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PMID:Separation of the adenovirus terminal protein precursor from its associated DNA polymerase: role of both proteins in the initiation of adenovirus DNA replication. 695 61

Enzyme kinetic measurements are presented showing that Km rather than maximum velocity (Vmax) discrimination governs the frequency of forming 2-aminopurine X cytosine base mispairs by DNA polymerase alpha. An in vitro system is used in which incorporation of dTMP or dCMP occurs opposite a template 2-aminopurine, and values for Km and Vmax are obtained. Results from a previous study in which dTTP and dCTP were competing simultaneously for insertion opposite 2-aminopurine indicated that dTMP is inserted 22 times more frequently than dCMP. We now report that the ratio of Km values KCm/KTm = 25 +/- 6, which agrees quantitatively with the dTMP/dCMP incorporation ratio obtained previously. We also report that VCmax is indistinguishable from VTmax. These Km and Vmax data are consistent with predictions from a model, the Km discrimination model, in which replication fidelity is determined by free energy differences between matched and mismatched base pairs. Central to this model is the prediction that the ratio of Km values for insertion of correct and incorrect nucleotides specifies the insertion fidelity, and the maximum velocities of insertion are the same for both nucleotides.
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PMID:Kinetic measurement of 2-aminopurine X cytosine and 2-aminopurine X thymine base pairs as a test of DNA polymerase fidelity mechanisms. 695 28

Chloroacetaldehyde, a rearranged metabolic product of the human carcinogen vinyl chloride, reacts with the DNA-like polymers poly(dA-dT) and poly(dC-dG) to form etheno-adducts of the adenine and cytosine bases. These treated polymers, when used as templates for E. coli DNA polymerase I in an in vitro assay, show a decreased ability to direct DNA synthesis. At the same time, increased relative levels of non-complementary nucleotides are incorporated. With the poly(dA-dT) templates 1 dGMP residue is incorporated for every approx 60 ethenoadenine residues present whilst no increased misincorporation of dCMP was detected. With the poly(dC-dG) templates 1 misincorporation of dAMP or dTMP occurred in the presence of approx 30 and 80 ethenocytosine residues respectively. A nearest neighbour analysis shows that with the modified poly(dC-dG) templates the majority of the errors were incorporated opposite cytosine (or modified cytosine) bases.
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PMID:The induction of errors during in vitro DNA synthesis following chloroacetaldehyde-treatment of poly(dA-dT) and poly(dC-dG) templates. 702 22

Bacteriophage XP-12-infected Xanthomonas oryzae have been found to be a source of a kinase preparation which converts m5dCMP to m5dCDP and then to m5dCTP using ATP as the phosphate donor. Optimal formation of the triphosphate required the presence of creatine phosphate and creatine kinase. In the presence of dGTP, dTTP and dATP, Escherichia coli DNA polymerase I and T4 DNA polymerase catalyzed the incorporation of m5dCTP into DNA just as efficiently as that of dCTP. Neither dTMP nor dCMP served as substrate for the m5dCMP monophosphate kinase. Analogous preparations from uninfected X. oryzae were unable to phosphorylate m5dCMP.
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PMID:A bacteriophage-induced 5-methyldeoxycytidine 5'-monophosphate kinase. 708 69

Hepadnaviruses employ a unique mechanism for the initiation of RNA-directed DNA synthesis. Initially, four bases (5'-GTAA-3') are added to a tyrosine residue of the viral polymerase by reverse transcription of a bulge sequence in epsilon, a stem-loop structure which functions as the packaging signal for pregenomic RNA. This protein-DNA complex acts as the primer for minus-strand elongation from the 3' sequence, DR1. To understand this process in greater detail, we investigated whether the protein-mediated priming of viral DNA synthesis is affected by nucleotide analogs. By using cell-free expression of duck hepatitis B virus (DHBV) reverse transcriptase (G.-H. Wang and C. Seeger, Cell 71:663-670, 1992), the 5'-triphosphate of the thymidine analog fialuridine (FIAU) was shown to inhibit the incorporation of radiolabeled TMP into primer DNA in a dose-dependent manner. Inhibition by the 5'-triphosphate of FIAU (FIAU-TP) was nearly complete at a concentration of 10 microM. The dideoxynucleotide analogs ddGTP, ddTTP, and 3'-azidodeoxythymidine triphosphate, known inhibitors of DHBV endogenous DNA polymerase, did not affect substantially the synthesis of primer DNA. Alternate substrate analysis suggested that FIAU is incorporated efficiently into nascent primer DNA as an analog of thymidine. Using site-directed mutagenesis to construct a mutant RNA template yielding a primer with the sequence 5'-GTAC-3', we demonstrated that FIAU-TP inhibited the incorporation of TMP, had no effect on that of dAMP, and decreased markedly the incorporation of dCMP. These results show that the synthesis of full-length DHBV primer DNA is inhibited by FIAU-TP but not by the dideoxynucleotide analogs that we tested. The significance of these findings as they relate to HBV DNA replication is discussed.
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PMID:Priming of duck hepatitis B virus reverse transcription in vitro: premature termination of primer DNA induced by the 5'-triphosphate of fialuridine. 752 86

3,N4-Etheno-2'-deoxycytidine, 3-(hydroxyethyl)-2'-deoxyuridine, and 3,N4-ethano-2'-deoxy-cytidine are found in DNA of cells treated with either vinyl chloride or 1,3-bis(2-chloroethyl)-nitrosourea. These exocyclic and related DNA adducts were incorporated into oligodeoxynucleotides, which were then used as templates for primer extension in reactions catalyzed by the Klenow fragment of Escherichia coli DNA polymerase I. The miscoding potential of each lesion was determined quantitatively. DNA primers were readily extended on an epsilon dC-modified template; dAMP and dTMP were incorporated opposite the lesion. With high concentrations of DNA polymerase, small amounts of fully extended reaction products containing dAMP and dGMP or one-base and two-base deletions opposite ethano-dC were formed. Primer extension was blocked partially on templates containing 3-(hydroxyethyl)-dU; dAMP and smaller amounts of dTMP and dCMP were incorporated. The frequencies of nucleotide insertion opposite each of the three lesions and the frequencies of chain extension from the 3'-primer terminus, determined by kinetic analysis, were consistent with results of experiments utilizing polyacrylamide gel electrophoresis. We conclude from these studies that epsilon dC, ethano-dC, and 3-(hydroxyethyl)-U are potentially miscoding lesions; only epsilon dC facilitates translesional synthesis.
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PMID:Miscoding by the exocyclic and related DNA adducts 3,N4-etheno-2'-deoxycytidine, 3,N4-ethano-2'-deoxycytidine, and 3-(2-hydroxyethyl)-2'-deoxyuridine. 770 60

A 25mer oligonucleotide containing a single N-(deoxyguanosin-8-yl-)-1-aminopyrene (dGAP), the major DNA adduct formed by reductively activated 1-nitropyrene, was synthesized. The adduct was located at nucleotide 21 from the 3' end. DNA synthesis on this template by human DNA polymerases alpha and beta, HIV reverse transcriptase, Sequenase (version 2.0) and Klenow fragment of DNA polymerase I was strongly blocked at the nucleotide 3' to the adduct site. Only when a 3'-->5' exonuclease-deficient Klenow fragment was used was incorporation of a nucleotide opposite the adduct observed. Nevertheless, extension beyond the adduct site did not occur to a significant extent. Only a relatively small proportion of full-length product (< 5%) was detected. In the presence of Mn2+, the efficiency of bypass with this polymerase increased. When a 20mer primer was elongated in the presence of only one nucleotide triphosphate, deoxycytidylic acid was preferentially incorporated opposite the adduct. Deoxycytidine opposite the adduct was also preferred when a set of 21mer primers (containing each of the four nucleotides opposite dGAP) were elongated to a full-length product in the presence of all four deoxynucleotide triphosphates. In order to confirm these results, extension of a 15mer primer was carried out with all four deoxynucleotide triphosphates and the products were isolated. Maxam--Gilbert sequencing of each elongation product showed that primer extension occurred in an error-free manner. We conclude that dGAP is a strong block of DNA replication. However, when translesion synthesis occurs, it is largely accurate.
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PMID:DNA polymerase action on an oligonucleotide containing a site-specifically located N-(deoxyguanosin-8-yl)-1-aminopyrene. 772 60

Mercurated DNA was synthesized in vitro by substituting Hg-dCTP or Hg-dUMP for dCTP in one strand of M13mp8DNA in a DNA polymerase I reaction. Restriction enzymes, including Sma I, Pst I, Bam HI, Hind III, and Hinc II, were completely inactive when their recognition sites were fully substituted with Hg-dCMP, while Hg-dUMP containing DNA was hydroxlyzed to some extent. Under conditions favoring star activities, or when DNA was substituted with a low level of mercury-nucleotide, DNA was cleaved by restriction enzymes. Susceptibility to degrading and synthesizing enzymes and insensitivity to restriction endonucleases of fully mercurated DNA makes mercuration an attractive molecular "tag" for in vitro manipulation and selective isolation of Hg-DNA.
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PMID:Effect of mercury substitution of DNA on its susceptibility to cleavage by restriction endonucleases. 774 94


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