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)

Even though Escherichia coli can grow in media containing up to 1 M NaCl, one-fifth that amount of NaCl will completely inhibit the in vitro activity of DNA polymerase III holoenzyme. It has been established that the major intracellular ionic osmolytes are potassium and glutamate (Richey, B., Cayley, D. S., Mossing, M. C., Kolka, C., Anderson, C. F., Farrar, T. C., and Record, M. T., Jr. (1987) J. Biol. Chem. 262, 7157-7164). We have found that holoenzyme catalyzes replication efficiently in vitro in up to 1 M potassium glutamate. Two salt effects on the replication of single-stranded DNA were observed. At low salt replicative activity was enhanced and at high salt there was anion-specific inhibition. We have found that DNA polymerase III holoenzyme tolerated 10-fold higher concentrations of glutamate than chloride. The ability of various anions to extend the useful range of salt concentrations followed the order: phosphate less than chloride less than N-Ac-glutamate less than acetate less than glycine less than aspartate less than glutamate. With the exception of phosphate, this order followed the Hofmeister series indicating that the anion-specific effects were due to anions interacting at the protein-water interface at weak anion binding sites. Glutamate did not reverse the inhibition by chloride. The low salt enhancement and high salt inhibition effects were additive for the two anions indicating that they competed for common anion binding sites. The major salt-sensitive step was holoenzyme binding to template rather than the subsequent elongation reaction.
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PMID:Glutamate overcomes the salt inhibition of DNA polymerase III holoenzyme. 256 34

An apparatus is described which permits the incubation of samples at three different temperatures in a cyclic fashion. The parts for the incubator are either present in every biochemical laboratory (water baths) or can be easily obtained at a low price (timers and magnetic valves). Thus the new DNA amplification procedure employing Thermus aquaticus-DNA polymerase can be carried out automatically without major investments.
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PMID:Low cost apparatus for primer-directed DNA amplification using Thermus aquaticus-DNA polymerase. 265 99

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

When cells are exposed to ionizing radiation, they suffer lethal damage (LD), potentially lethal damage (PLD), and sublethal damage (SLD). All three forms of damage may be caused by direct or indirect radiation action or by the interaction of indirect radiation products with direct DNA damage. In this report I examine the expression of LD and PLD caused by the indirect action of X rays in isogenic, repair-deficient Escherichia coli. The radiosensitivity of a recA mutant, deficient both in pre- and post replication recombination repair and SOS induction (inducible error-prone repair), was compared to that of a recB mutant which is recombination deficient but SOS proficient and to a previously studied DNA polymerase 1-deficient mutant (polA) which lacks the excision repair pathway. Indirect damage by water radicals (primarily OH radicals) was circumvented by the presence of 2 M glycerol during irradiation. Indirect X-ray damage by water radicals accounts for at least 85% of the PLD found in exposed repair-deficient cells. The DNA polymerase 1-deficient mutant is most sensitive to indirect damage with the order of sensitivity polA1 greater than recB greater than or equal to recA greater than wild type. For the direct effects of X rays the order of sensitivity is recA greater than recB greater than polA1 greater than wild type. The significance of the various repair pathways in mitigating PLD by direct and indirect damage is discussed.
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PMID:Free radical scavenging and the expression of potentially lethal damage in X-irradiated repair-deficient Escherichia coli. 281 36

Foscarnet (trisodium phosphonoformate) is a new antiviral compound with in vitro inhibitory effects against the DNA polymerases of hepadna viruses. To study the effects of the drug in chronic hepadna virus infection, we treated ducks chronically infected with duck hepatitis B virus for 10 days with either low-dose foscarnet (50 mg/kg i.p. b.i.d.), high-dose foscarnet (250 mg/kg i.p. b.i.d.), or sterile water injections. Serum duck hepatitis B virus DNA and intrahepatic replicative forms of the virus were measured using molecular biological techniques with both a double-stranded radiolabeled DNA probe and a plus-strand (noncoding) specific RNA probe. We found a dose-related decrease in serum and intrahepatic duck hepatitis B virus DNA during treatment, with a rapid return toward baseline values after the cessation of treatment. There was a disproportionate decrease in the plus strand of viral DNA with treatment. We conclude that foscarnet exerts its effect in hepadna virus infection through inhibition of viral DNA polymerase. Further study is necessary to determine whether foscarnet, by itself or in combination with other treatment modalities, has a role to play in the treatment of chronic hepatitis B infections in humans.
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PMID:Foscarnet decreases serum and liver duck hepatitis B virus DNA in chronically infected ducks. 294 28

An automated system is described that performs the cyclic temperature changes required for enzymatic amplification of specific DNA segments in vitro using the polymerase chain reaction (pcr). During pcr, oligonucleotide primer molecules are bound at low temperature to templates of heat-denatured DNA and extended on their 3' end using a thermostable DNA polymerase. The DNA denaturation, primer annealing, and extension is repeated several times under program control to accumulate a large number of identical copies of the DNA sequence between the primers. A microcomputer system controls the flow of 96 degrees C and 37 degrees C water through a 24-well sample holder so that the temperature in the samples in the holder varies as required for DNA denaturation, primer annealing, and DNA polymerization. The microcomputer automatically performs multiple thermal cycles and is sufficiently flexible that the temperature profile can be varied from cycle to cycle.
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PMID:A programmable system to perform the polymerase chain reaction. 320

This study was designed to investigate the role of ornithine decarboxylase (ODC) and polyamines in pancreatic adaptation. Cholecystokinin (CCK) is well-known to be a potent trophic stimulus on the pancreas. On the other hand, the oral application of the synthetic trypsin inhibitor camostate results in an extensive release of endogenous CCK in rats. alpha-difluoromethylornithine (DFMO), an irreversible and specific inhibitor of ODC, was applied simultaneously to elucidate the essential role of polyamines in pancreatic growth. Camostate feeding (200 mg/kg b.wt. orally twice a day) resulted in a rapid elevation of ODC activity already after 2 hours, reaching a maximum after 6 hours (about 200fold above controls) followed by a significant increase in putrescine after 4 hours and spermidine after 24 hours while spermine remained unchanged. The trophic parameters increased as expected in following time-course: thymidine kinase (12 hours), DNA polymerase (12 hours), protein (24 hours), pancreatic weight (24 hours) and DNA (5 days). DFMO (2% in drinking water + 3 x 300 mg/kg b.wt. i.p. during daytime) was not able to prevent but significantly delayed and reduced the camostate-induced increase in ODC and polyamines as well as the trophic parameters. These data indicate an essential role for ODC and polyamines in camostate-induced pancreatic growth and hormonal mediated pancreatic adaptation.
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PMID:Ornithine decarboxylase and polyamine biosynthesis in pancreatic adaptation. 325 34

The purine base and nucleoside analogues N2-(p-n-butylphenyl)-guanine (BuPh-Gua) and N2-(p-n-butylphenyl)-2'-deoxyguanosine (BuPh-dGuo) are strong inhibitors of isolated mammalian DNA polymerase alpha, but are less potent that expected as inhibitors of DNA replication in intact cultured cells [G. E. Wright, L. W. Dudycz, Z. Kazimierczuk, N. C. Brown and N. N. Khan, J. med. Chem. 30, 109 (1987)]. The mechanistic basis for these observations was explored using permeable human fibroblasts. DNA replication in the permeable cells was inhibited only slightly by BuPh-Gua and BuPh-dGuo at 100 microM, the highest concentration which could be attained. Similar results were obtained for ultraviolet-induced DNA repair synthesis, a process which is though to involve the same DNA polymerase as replication. More detailed studies were performed using the corresponding nucleotide analogue, N2-(p-n-butylphenyl)-2'-deoxyguanosine-5'-triphosphate (BuPh-dGTP), which is much more water-soluble than the base and nucleoside. The apparent Ki values for BuPh-dGTP inhibition of both replication and ultraviolet-induced repair synthesis in permeable cells were approximately 3 microM. These values are several hundred-fold greater than the apparent Ki for BuPh-dGTP inhibition of isolated human DNA polymerase alpha, which is approximately 10 nM. We conclude that BuPh-Gua and BuPh-dGuo are poor inhibitors of DNA replication in intact cells not because of permeability barriers, but because, unlike polymerase alpha, cellular DNA synthesis is relatively insensitive to this group of inhibitors. These results suggest that polymerase alpha may not be a good general model for predicting the potency of base, deoxyribonucleoside and deoxyribonucleotide analogues as inhibitors of mammalian cellular DNA replication. The fact that the permeable cell systems accurately reflect the relative insensitivity to butylphenyl-guanine derivatives of mammalian DNA replication suggests that permeable cells may be useful tools in future studies of base and nucleoside analogues.
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PMID:Analysis of butylphenyl-guanine, butylphenyl-deoxyguanosine, and butylphenyl-deoxyguanosine triphosphate inhibition of DNA replication and ultraviolet-induced DNA repair synthesis using permeable human fibroblasts. 335 81

The present experiments were conducted to determine the effects of cyclophosphamide (150 mg/kg) on the pathophysiology of RIF-1 solid tumors and to determine the temporal relationship between treatment mediated changes in tumor vascular physiology, cell proliferation, and chemoresponsiveness in vivo. Capillary permeability and plasma and extracellular water volumes were determined by a 125I-bovine serum albumin, 51Cr-EDTA double isotope dilution assay at various intervals after cyclophosphamide. Tumor blood flow and exchangeable erythrocyte vascular volumes were determined by 86RbCl distribution and 51Cr-labeled erythrocyte dilution methods. Cell proliferation in RIF-1 tumors, assessed by [3H]thymidine labeling index and tumor growth fraction (primer-dependent DNA polymerase labeling assay) measurements, was inhibited for up to 3 days by cyclophosphamide. Although tumor regrowth was not apparent until Day 10, cell kinetic studies indicated proliferative recovery in the surviving cell population on Days 4 and 5 after treatment. Increases in tumor blood flow and tumor vascular volumes were temporally coincident with this proliferative response. In split-dose experiments, the time-dependent increases in the chemoresponsiveness of RIF-1 tumors, after cyclophosphamide, may be due not only to the increased proliferation of repopulating cells, but also to vascular responses attendant with cytoreduction.
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PMID:Effect of cyclophosphamide on the pathophysiology of RIF-1 solid tumors. 339 Aug 14

The relation between DNA polymerase fidelity and base pairing stability is investigated by using DNA primer-template duplexes that contain a common 9-base template sequence but have either correct (A.T) or incorrect (G.T, C.T, T.T) base pairs at the primer 3' terminus. Thermal melting and enzyme kinetic measurements are compared for each kind of terminus. Analysis of melting temperatures finds that differences between the free energy changes upon dissociation (delta delta Go) are only 0.2, 0.3, and 0.4 kcal.mol-1 (1 cal = 4.18 J) for terminal A.T compared to G.T, C.T, and T.T mispairs, respectively, at 37 degrees C. We show that enthalpy changes are directly correlated with entropy changes for normal and abnormal base pairs in DNA in aqueous solution and that delta delta Go values are small because of near cancellation of corresponding enthalpy and entropy components. The kinetics of elongating primer termini are measured with purified Drosophila DNA polymerase alpha. The matched A.T terminus is found to be extended approximately 200 times faster than a G.T mismatch and 1400 and 2500 times faster than C.T and T.T mismatches, respectively. Enzymatic discrimination against elongating mismatched termini is based mainly on Km rather than Vmax differences. From Km at 37 degrees C, we find delta delta Go values of 2.6-3.7 kcal.mol-1, about an order of magnitude greater than indicated by melting data. A similar measurement of nucleotide insertion kinetics has previously found rates of forming A.T base pairs to be 500 times greater than G.T mispairs and 20,000 times greater than C.T and T.T mispairs. Here also, Km differences are mainly responsible for discrimination and indicate even larger delta delta Go values (4.3-4.9 kcal.mol-1). Thus, free energy differences between correct and incorrect base pairs in the active site cleft of polymerase appear to be greater than 10 times as large as in aqueous medium. We explore the idea that a binding cleft that snugly fits correct base pairs and excludes water at the active site may amplify base-pair free energy differences by reducing entropy differences and increasing enthalpy differences sufficiently to account for nucleotide insertion and extension fidelity.
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PMID:Comparison between DNA melting thermodynamics and DNA polymerase fidelity. 341 95


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