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)

A simple acetoxymercuration reaction for introducing covalently bound mercury atoms into nucleotides is described. The 5-mercuriacetate derivatives of UTP, CTP, dUTP, and dCTP, as well as the 7-mercuriacetate derivative of 7-deazaATP, have been prepared by this procedure and tested as substrates for nucleic acid polymerases. These nucleotides, in the absence of added mercaptan, are not polymerized and in most instances are potent enzyme inhibitors. However, conversion of these mercuriacetates to mercurithio compounds in situ by the addition of one of various mercaptans, yields nucleoside triphosphates that are excellent substrates for all polymerases tested: Escherichia coli and T7 RNA polymerases, DNA polymerase I of E. coli, DNA polymerase of avian myeloblastosis virus, and calf-thymus terminal deoxynucleotidyl transferase. By varying the mercaptan used to promote syntheses it is possible to access certain structural limitations in the enzyme's nucleoside triphosphate binding site. These mercurinucleotides appear to have a diversity of potential applications: (1) as heavy-atom reagents for crystallographic and microscopic studies; (2) as affinity probes for enzymes sensitive to sulfhydryl modification; (3) as steric probes of substrate-binding sites on enzymes; and (4) as reagents for forming covalent protein-polynucleotide complexes.
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PMID:The synthesis and enzymatic polymerization of nucleotides containing mercury: potential tools for nucleic acid sequencing and structural analysis. 436 67

Herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) DNA polymerases were highly purified from infected HeLa BU cells by DEAE cellulose, phosphocellulose and DNA cellulose column chromatography. DNA exonuclease activity but not endonuclease activity was found associated with both types of DNA polymerase. Both DNA polymerase activities could be activated by salt in a similar fashion with the optimal activity in the range of ionic strength between 0.22 and 0.29 alpha. At an ionic strength of 0.14, spermidine and putrescine in the concentration range (0--5 mM) studied could mimic the action of KCI in stimulating DNA polymerase activity. Spermine, in the same concentration range, had a biphasic effect. At an ionic strength of 0.29 all three polyamines were inhibitory. HSV-1 and HSV-2 DNA polymerase are similar in their column chromatographic behavior, sedimentation rate in sucrose gradient centrifugation, and activation energy, but they differ in their heat stability at 45 degrees C with the HSV-2 enzyme more stable than the HSV-1 enzyme. Kinetic behavior of both enzymes is similar, with Km values for deoxyribonucleoside triphosphates in the range of 5 . 10(-7) to 1.8 . 10(-8) M. IdUTP and dUTP served as apparent competitive inhibitors with respect to dTTP, and AraATP acted as an apparent competitive inhibitor with respect to dATP. AraATP could not replace dATP in the DNA polymerization reaction; in contrast, IdUTP could replace TTP. Phosphonoformic acid behaved as an uncompetitive inhibitor with respect to DNA. The ID(50) value estimated was foind to be dependent on the purity of the DNA polymerase used and the ionic strength of the assay condition. Each DNA-polymerase associated DNA exonuclease had the same stability at 45 degrees C as its DNA polymerase. The associated DNAase activity was inhibited by phosphonoformic acid and high ionic strength of the assay condition.
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PMID:Properties of herpes simplex virus type 1 and type 2 DNA polymerase. 625 Jun 18

Several triphosphates (TP) of 5-substituted deoxyuridine (dU), like 5-ethyl (Et), 5-n-propyl (n-Pr), 5-iso-propyl (iso-Pr), 5-n-hexyl (n-Hx), and 5-trifluorothymidine (F3-dT) were used as substrates for HeLa DNA polymerase alpha and for two herpes simplex virus (HSV)-coded DNA polymerases isolated from HeLa cells infected with HSV-1, strain C42 (wild-type), or its mutant resistant to phosphonoformate (PFAr). All polymerases were purified up to the DNA-cellulose column step and they showed comparable specific activities. The incorporation into DNA studied with all the alkyl analogues of dUTP is several times higher with the virus enzymes than with DNA polymerase alpha. The DNA polymerase of the mutant virus incorporates dUTP analogues to a lower extent than the wild-type polymerase. The two virus enzymes also differ in the Km and Vmax values for different substrates, indicating that the mutation to PFAr has affected the structure of the virus DNA polymerase. Surprisingly, all three enzymes use F3-dTTP as substrate for DNA synthesis to an equal but limited extent.
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PMID:Differential incorporation of thymidylate analogues into DNA by DNA polymerase alpha and by DNA polymerases specified by two herpes simplex viruses. 629 May 94

3'-NH2-BV-dUrd, the 3'-amino derivative of (E)-5-(2-bromovinyl)-2'-deoxyuridine, was found to be a potent and selective inhibitor of herpes simplex virus type 1 (HSV-1) and varicella-zoster virus (VZV) replication. 3'-NH2-BV-dUrd was about 4-12 times less potent but equally selective in its anti-herpes activity as BV-dUrd. Akin to BV-dUrd, 3'-NH2-BV-dUrd was much less inhibitory to herpes simplex virus type 2 than type 1. It was totally inactive against a thymidine kinase-deficient mutant of HSV-1. The 5'-triphosphate of 3'-NH2-BV-dUrd (3'-NH2-BV-dUTP) was evaluated for its inhibitory effects on purified herpes viral and cellular DNA polymerases. Among the DNA polymerases tested, HSV-1 DNA polymerase and DNA polymerase alpha were the most sensitive to inhibition by 3'-NH2-BV-dUTP (Ki values 0.13 and 0.10 microM, respectively). The Km/Ki ratio for DNA polymerase alpha was 47, as compared with 4.6 for HSV-1 DNA polymerase. Thus, the selectivity of 3'-NH2-BV-dUrd as an anti-herpes agent cannot be ascribed to a discriminative effect of its 5'-triphosphate at the DNA polymerase level. This selectivity most probably resides at the thymidine kinase level. 3'-NH2-BV-dUrd would be phosphorylated preferentially by the HSV-1-induced thymidine kinase (Ki 1.9 microM, as compared with greater than 200 microM for the cellular thymidine kinase), and this preferential phosphorylation would confine the further action of the compound to the virus-infected cell.
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PMID:Antiviral activity of the 3'-amino derivative of (E)-5-(2-bromovinyl)-2'-deoxyuridine. 630 80

The physical and biochemical properties of two pairs of synthetic DNA template-primers were investigated. The copolymer poly(dA-dU) . poly(dA-dU) and the homopolymer duplex poly(dA). poly(dU) were characterized by a lower Tm and by a higher buoyant density value than the respective thymine polynucleotides poly(dA-dT) . poly(dA-dT) and poly(dA) . poly(dT). The polymerizing and the primer terminus adding reactions of a homogenous E. coli DNA polymerase I preparation, as measured by incorporation of [3H]dAMP into the acid-insoluble fraction, were significantly poorer with uracil-containing template-primers than with thymine templates. Moreover, the uracil-containing polynucleotides inhibited the polymerizing activity of DNA polymerase I to a greater extent than the thymine polynucleotides, when the enzymatic activity was investigated with a dATP/dTTP/dUTP-free incorporation system making use of poly(dI-dC) . poly(dI-dC) as the template-primer.
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PMID:Uracil in deoxyribonucleotide polymers reduces their template-primer activity for E. coli DNA polymerase I. 634 14

(E)-5-(2-bromovinyl)-2'-deoxyuridine 5'-triphosphate (BVdUTP), known as a specific inhibitor of herpes simplex virus (type 1)-DNA polymerase, was found to be a potent inhibitor of the activity of terminal deoxynucleotidyltransferase (TdT) from calf thymus. BVdUTP was not an efficient substrate of TdT, but it inhibited the incorporation of normal deoxynucleotide substrates in competitive fashion at the nucleotide binding site of TdT molecule. The Ki value for BVdUTP (5 microM) was much less than the Km value for dGTP (83 microM), indicating stronger affinity of the inhibitor to TdT than that of the substrate. These results indicate the usefulness of BVdUTP as a potent inhibitor of TdT for elucidation of the reaction mechanism of this enzyme.
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PMID:Inhibition of terminal deoxynucleotidyltransferase by (E)-5-(2-bromovinyl)-2'-deoxyuridine 5'-triphosphate. 666 44

DNA polymerases from procaryotic sources can utilize a variety of dTTP analogues as substrates. We studied here in vitro DNA syntheses catalyzed by DNA polymerase alpha and beta of calf thymus, and for comparison, by the Escherichia coli DNA polymerase I large fragment enzyme in the presence of 5-alkyl derivatives of dUTP as dTTP substrate analogues, using activated DNA as template-primer. The alkyl substituents were n-alkyl (from ethyl to hexyl) and iso-alkyl (isopropyl and tert-butyl) groups. All enzymes were active in the presence of each modified dTTP, incorporation rates of [3H]dAMP or [3H]dGMP were, however, much lower with the analogues than with dTTP. According to relative incorporation rates, alpha-polymerase in DNA synthesis was found to be less sensitive to changes in the length of the alkyl substituent of 5-n-alkyl-dUTPs than beta-polymerase or the E. coli enzyme. Evidence for the incorporation of the analogues was presented for 5-[2-14C]isopropyl-dUTP.
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PMID:A study of substrate specificity of mammalian and bacterial DNA polymerases with 5-alkyl-2'-deoxyuridine 5'-triphosphates. 698 14

1. The incorporation and excision kinetics of Escherichia coli DNA polymerase I in the replication of (dA)n with dUTP and (dI)n with dCTP was studied at various temperatures and pH. When the incorporation/excision ratio (dy/dx) is plotted against the concentration of deoxynucleoside triphosphate [S] two kinds of curves are obtained. With (dI)n, dy/dx increases with [S], then reaches an asymptotic value. This behaviour, consistent with a kinetic amplification or kinetic proofreading mechanism, is observed at all temperatures and pH. With (dA)n, dy/dx increases with [S] but in a convex, instead of a concave manner. In this case, we approximated the curves by straight lines at the origin, in conformity with the prediction of the frayed-unfrayed model. Again a single behaviour is consistently observed at all temperatures and pH. 2. The data were analyzed in terms of ratios or products of three kinetic constants: ki for incorporation, ke for excision and, in the (dI)n system, theta for the processing of the incoming dNTP. The dNMP production in the (dI)n system is dominated by the ke theta term which increases with temperature and pH. Temperature influences excision more than incorporation, the net result being a linear decrease of ki/ke with temperature. The effect is more pronounced with (dI)n than with (dA)n and is probably related to the stability of the template-primer complex. The ki/ke term shows a bell-shaped dependency with pH in the (dI)n system. With (dA)n it remains constant between pH 7.5 and 8.5 then decreases with a transition midpoint at pH 9.0. We suggest that the pH profiles may reflect the ionization of the template in the first case, and of the substrate in the second.
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PMID:Polymerization/excision kinetics of Escherichia coli DNA polymerase I. Stability in kinetic behaviour and variations of the rate constants with temperature and pH. 704 74

The nick translation procedure without external addition of nicking enzymes was performed in situ on fixed nuclei of mouse preimplantation, and postimplantation embryos, as well as bone marrow in order to detect possible DNA single-strand breaks. All preparations of nuclei were made using the same technique. Nick translation of nuclear DNA with DNA polymerase I in the presence of biotinylated-dUTP demonstrated a characteristic absence of label on nuclei of postimplantation embryos and bone marrow. The nuclear reactivity varied according to the cleavage divisions of the zygote, being highest at the four-cell stage.
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PMID:Single-strand DNA breaks in nuclei of early mouse embryos detected by in situ nick translation. 774 62

The nick translation and gap filling procedures, without external addition of nicking enzymes, were performed in situ on fixed chromosomes of mouse preimplantation and postimplantation embryos and of bone marrow in order to detect possible DNA single-strand breaks (nicks and (or) gaps). All chromosome preparations were made using the same technique. Nick translation of chromosomal DNA with DNA polymerase I (Pol I) or gap filling with the Klenow fragment of Pol I in the presence of biotinylated-dUTP, demonstrated a regular absence of label on chromosomes of postimplantation embryos and bone marrow. No difference in sensitivity was found between the holoenzyme and the Klenow fragment. In preimplantation embryos, the chromosome reactivity in nick translation was highest at the blastocyst stage and varied according to cleavage divisions of the zygote.
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PMID:DNA-strand breaks in chromosomes of early mouse embryos as detected by in situ nick translation and gap filling. 777 4


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