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 propose a model for DNA polymerase fidelity in which free energy differences, delta delta G, between matched and mismatched nucleotides are magnified at the enzyme's active site. Both hydrogen bonding and stacking components of the interaction energy are amplified, with the most profound effect being on the magnitude of hydrogen-bonding interactions. Magnification in delta delta G values follows from the exclusion of water around base pairs in the active site cleft of the enzyme. After showing that base-pair dissociation energies calculated from hydrogen-bonding and base-stacking interactions in vacuo are greatly reduced by water, it is proposed that water removal results in a proportional restoration of these contributions to base pairing. Assuming approximately equal to 40% exclusion of surrounding water, one predicts magnified values of delta delta G sufficient to account for polymerase insertion and proofreading fidelity, thereby avoiding the need to postulate additional active site constraints in order to select or reject nucleotides.
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PMID:Comparison of nucleotide interactions in water, proteins, and vacuum: model for DNA polymerase fidelity. 345

Kinetics of the synthesis of adducts between salmon testis DNA and platinum(II) compounds were measured by their effects on DNA synthesis, circular dichroism, and ethidium bromide dependent fluorescence. Transient incorporation of [14C]cyanide into DNA adducts of of cis-diammineaquochloroplatinum(II) and respectively cis-diamminediaquoplatinum(II) compounds but not of trans-diammineaquochlorplatinum(II) was observed. A minimal kinetic scheme is derived, in which a transient monodentate DNA-platinum(II) adduct is formed in a bimolecular reaction between DNA and aquated platinum(II) compounds. Second-order rate constants are 2000-3000 M-1 min-1 for cis-diamminediaquoplatinum(II) and 280-400 M-1 min-1 for cis- and trans-diammineaquochloroplatinum(II), respectively. The dependence of pseudo-first-order rate constants is not linear for high concentrations of DNA, suggesting competitive formation of more than one primary adduct. The monodentate adducts inhibit DNA polymerase catalyzed DNA synthesis. The biomolecular reaction is followed by a rearrangement (rate constant 0.22 min-1) that gives rise to most of the decrease in the fluorescence intensity and that depends on the state of aquation of the DNA-bound platinum(II) complex. By exchange of coordinated water with a second nucleotide, the monodentate adduct can form cross-links in a reaction joining the rearrangement. Adducts containing a chloro group liberate it by hydrolysis prior to cross-linking. In the case of the trans-platinum(II) adduct, the hydrolysis is aided by the trans effect of the bound first nucleotide.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Kinetic investigation of the DNA platination reaction: evidence for a transient adduct between deoxyribonucleic acid and cis-platinum(II). 355 39

The purpose of this study was to characterize the ternary complexes formed in the reaction of cis-diamminedichloroplatinum (II) (cis-DDP) and nucleic acids, in the presence of the intercalating compound ethidium bromide (EtBr). In these ternary complexes, some EtBr is tightly bound to the nucleic acids. Tight binding is defined by resistance to extraction with butanol, assayed by filtration at acid pH or thin layer chromatography at basic pH. These ternary complexes are formed with double stranded but not with single stranded nucleic acids. They are not formed if cis-DDP is replaced by transdiamminedichloroplatinum(II). The amount of tightly bound EtBr depends upon the sequence of the nucleic acid, being larger with poly (dG-dC).poly(dG-dC) than with poly(dG).poly(dC). Spectroscopic results support the hypothesis that the tight binding of the dye is due to the formation of a bidentate adduct (guanine-EtBr)cis-platin. The visible spectrum of the ternary complexes is blue-shifted as compared to that of EtBr intercalated between the base pairs of unplatinated DNA and it depends upon the conformation of the ternary complex. The fluorescence quantum yield of the ternary complexes is lower than that of free EtBr in water. Tightly bound EtBr stabilizes strongly the B form versus the Z form of the ternary complex poly(dG-dC)-Pt-EtBr and slows down the transition from the B form towards the Z form. The sequence specificity of cis-DDP binding to a DNA restriction fragment in the absence or presence of EtBr is mapped by means of the 3'----5' exonuclease activity of T4 DNA polymerase. In the absence of the dye, all the d(GpG) sites and all the d(ApG) sites but one in the sequence d(TpGpApGpC) are platinated. The d(GpA) sites are not platinated. In the presence of EtBr, some new sites are detected. These results might help to explain the synergism for drugs used in combination with cis-DDP and in the design of new chemotherapeutic agents.
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PMID:Characterization of the ternary complexes formed in the reaction of cis-diamminedichloroplatinum (II), ethidium bromide and nucleic acids. 382 39

(Sp)-2'-Deoxyadenosine 5'-O-[1-17O,1-18O,1,2-18O]triphosphate has been synthesized by desulfurization of (Sp)-2'-deoxyadenosine 5'-O-(1-thio[1,1-18O2]diphosphate) with N-bromosuccinimide in [17O]water, followed by phosphorylation with phosphoenolpyruvate-pyruvate kinase. A careful characterization of the product using high-resolution 31P NMR revealed that the desulfurization reaction proceeded with approximately 88% direct in-line attack at the alpha-phosphorus and 12% participation by the beta-phosphate to form a cyclic alpha,beta-diphosphate. The latter intermediate underwent hydrolysis by a predominant nucleophilic attack on the beta-phosphate. This complexity of the desulfurization reaction, however, does not affect the stereochemical integrity of the product but rather causes a minor dilution with nonchiral species. The usefulness of the (Sp)-2'-deoxyadenosine 5'-O-[1-17O,1-18O,1,2-18O]triphosphate in determining the stereochemical course of deoxyribonucleotidyl-transfer enzymes is demonstrated by using it to delineate the stereochemical course of the 3'----5'-exonuclease activity of DNA polymerase I. Upon incubation of this oxygen-chiral substrate with Klenow fragment of DNA polymerase I in the presence of poly[d(A-T)] and Mg2+, a quantitative conversion into 2'-deoxyadenosine 5'-O-[16O,17O,18O]monophosphate was observed. The stereochemistry of this product was determined to be Rp. Since the overall template-primer-dependent conversion of a deoxynucleoside triphosphate into the deoxynucleoside monophosphate involves incorporation into the polymer followed by excision by the 3'----5'-exonuclease activity and since the stereochemical course of the incorporation reaction is known to be inversion, it can be concluded that the stereochemical course of the 3'----5'-exonuclease is also inversion.
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PMID:Stereochemical course of the 3'----5'-exonuclease activity of DNA polymerase I. 609 2

Seven different test systems were utilized to investigate the genetic activity of chromium compounds: infidelity of DNA replication in vitro by DNA pol alpha from calf thymus, damage of DNA detected by alkaline elution in treated mammalian cells or in DNA purified and treated in vitro, DNA repair synthesis in mammalian cells in vitro detected by autoradiography or scintillation counting after labelling with [3H]dThd, gene mutations in the Salmonella typhimurium Ames test, gene mutations (6TG resistance) in cultured hamster cells, sister-chromatid exchanges in different rodent cell cultures, and transformation to anchorage-independent growth of hamster cells in vitro (soft-agar assay). Potassium dichromate and chromium chloride were used as water-soluble Cr(VI) and Cr(III) salts. Several reference mutagens (EMS, MMS, MMC, 4NQO) were included in the single tests as positive controls. Cr(VI) was active in all the tested systems, except in the induction of DNA damage and DNA repair synthesis in cultured cells. Cr(III), on the other hand, was absolutely inactive unless a direct interaction with purified DNA was permitted by the test conditions. The relevance of data from the various tests to the understanding of the mechanisms of the genotoxic activity of chromium is discussed. Effects other than the direct interaction of Cr(III) with DNA are inferred, which can cause infidelity of the DNA polymerase functions.
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PMID:Genetic effects of chromium compounds. 634 55

A preparation of bacteriophage T4-induced deoxyribonucleotide synthetase complex is described. This very large complex of enzymes can be separated by centrifugation at 100,000 X g, by sucrose step gradient centrifugation, or with molecular exclusion columns. By direct assay and by unidimensional and two-dimensional acrylamide electrophoretic separations the following T4-coded enzymes were shown to be associated with the complex: ribonucleoside diphosphate reductase, dCMP deaminase, dCTP/dUTPase, dCMP hydroxymethylase, dTMP synthetase, and DNA polymerase. Other phage-coded prereplicative proteins related to DNA replication and other phage functions such as the proteins coded by genes 32, 46, rIIA, and rIIB as well as many unidentified proteins were also consistently associated with the isolated fractions. T4 DNA topoisomerase, a membrane-bound enzyme, was found in quantity in all purified fractions of the complex, even in preparations apparently free of membrane and of T4 DNA. The functional integrity of a segment of the complex was followed by measuring the conversion of [5-3H]CDP to the level of 5-hydroxymethyl dCMP. This series of reactions requires the actions of T4-coded ribonucleoside diphosphate reductase and its associated reducing system, dCTP/dUTPase and dCMP hydroxymethylase, 3H being lost to water at the last step. In this reaction sequence an intermediate, [5-3H]dCMP, is maintained at low steady state concentrations, and argument is presented that the synthesis of deoxyribonucleotides is channeled and normally tightly coupled to DNA replication. One of the primary characteristics of this complex is its ready dissociation of dilution into smaller complexes of proteins and to the free forms of the proteins. That the complex is held together by weak electrostatic forces was supported by its sensitivity to dissociation at moderate salt concentrations. Not only the enzymes required in deoxyribonucleotide synthesis but T4 DNA polymerase, T4 DNA topoisomerase, and a number of other proteins dissociate to varying degrees from the larger complexes under these conditions.
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PMID:Characteristics of a bacteriophage T4-induced complex synthesizing deoxyribonucleotides. 675 52

Earlier studies have shown guanine arabinoside (ara-G) is an effective agent against growth of T-cell lines and freshly isolated human T-leukemic cells. However, poor water solubility of ara-G limits clinical use. 2-Amino-6-methoxypurine arabinoside (506U) is a water-soluble prodrug converted to ara-G by adenosine deaminase. 506U is not a substrate for deoxycytidine kinase, adenosine kinase, or purine nucleoside phosphorylase and is phosphorylated by mitochondrial deoxyguanosine kinase at a rate 4% that of ara-G phosphorylation. Mitochondrial DNA polymerase was the least sensitive to ara-GTP inhibition of the five human DNA polymerases tested. [3H]506U was anabolized to ara-G 5'-phosphates in CEM cells but not to phosphorylated metabolites of 506U. 506U was selective for transformed T over B cells and also inhibited growth in two of three monocytic lines tested. 506U given i.v. to cynomolgus monkeys was rapidly converted to ara-G; the ara-G had a half-life of approximately 2 h. 506U had in vivo dose-dependent efficacy against human T-cell tumors in immunodeficient mice. A Phase 1 trial of 506U against refractory hematological malignancies is now in progress at two study sites.
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PMID:2-Amino-6-methoxypurine arabinoside: an agent for T-cell malignancies. 761 70

In this study, we have developed a chemically sensitive and specific polymerase chain reaction (PCR) assay to detect the presence of Streptococcus pneumoniae genomic DNA. The target DNA sequence was a 322-base pair segment of the S. pneumoniae DNA polymerase I gene (pol I). PCR products of pure cultures of a set of pneumococcal serotypes commonly associated with human infection could be amplified in water and in blood cultures of clinical isolates containing S. pneumoniae. We were able to detect 2 fg of purified S. pneumoniae DNA. There were no false-positive reactions when the assay was performed on samples containing the following clinically encountered bacteria: Haemophilus influenzae type B, Neisseria meningitidis, Escherichia coli, Klebsiella pneumoniae, Pseudomonas spp. nontypeable H. influenzae, Staphylococcus aureus, coagulase-negative staphylococci, and Streptococcus pyogenes. The addition of EDTA and citrate-anticoagulated whole blood to the PCR reaction mixture inhibited the PCR assay, whereas the addition of lithium heparin, sodium heparin, and sodium polyanetholesulfonate-anticoagulated whole blood to PCR reaction mixture did not interfere with the ability to detect the presence of S. pneumoniae DNA.
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PMID:Development of a polymerase chain reaction assay to detect the presence of Streptococcus pneumoniae DNA. 770 31

We investigate enthalpy-entropy compensation for melting of nearest-neighbor doublets in DNA. Based on data for 10 normal doublets and for doublets containing a mispaired or analog base, the correlation of delta Szero with delta Hzero follows a rectangular hyperbola. Doublet melting temperature relates linearly to delta Hzero by Tm = T(o) + delta Hzero/a, where T(o) = 273 K and a = 80 cal/mol-K. Thus Tm is proportional to delta Hzero + aTo rather than to delta Hzero alone as previously thought by assuming delta Szero to be constant. The term aTo = 21.8 kcal/mol may reflect a constant enthalpy change in solvent accompanying the DNA enthalpy change for doublet melting and is roughly equivalent to breaking four H-bonds between water molecules for each melted doublet. The solvent entropy change (aTo/Tm) declines with increasing Tm, while the DNA entropy change (delta Hzero/Tm) rises, so the combined DNA + solvent entropy change stays constant at 80 cal/K/mol of doublet. If such constancy in DNA + solvent entropy changes also holds for enzyme clefts as "solvent," then free energy differences for competing correct and incorrect base pairs in polymerase clefts may be as large as enthalpy differences and possibly sufficient to account for DNA polymerase accuracy. The hyperbolic relationship between delta Szero and delta Hzero observed in 1 M salt can be used to evaluate delta Hzero and delta Szero from Tm at lower, physiologically relevant, salt concentrations.
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PMID:Enthalpy-entropy compensation in DNA melting thermodynamics. 782 5

We have examined the biophysical properties of DNA polymerase beta (beta-pol) in solution. Time-resolved and steady-state fluorescence were used to investigate the microenvironment of the lone tryptophanyl residue (Trp324), and a combination of sedimentation equilibrium, sedimentation velocity and fluorescence anisotropy decay measurements were used to study the hydrodynamic properties of the enzyme. Trp324 appears to be exposed to water as judged by the tryptophan emission and steady-state and lifetime quenching experiments. The fluorescence is easily quenched by a neutral quencher acrylamide (kq = 1.59 x 10(9)M-1S-1), and by a negatively charged ionic quencher, I- (kq = 1.60 x 10(9) M-1S-1), but not by a positively charged ionic quencher, Cs+ (kq = 0.2 x 10(9) M-1S-1). The fluorescence lifetime of beta-pol is best described by the sum of two exponentials with a longer lifetime component of 8.4 ns and a shorter lifetime component of 1.3 ns. Decay associated spectra (DAS) show emission maxima at 340 nm and at 345 nm for the shorter lifetime and longer lifetime components, respectively, with corresponding centers of gravity at 347 nm and 348 nm. Sedimentation equilibrium experiments show that the enzyme exists as a monomer at the KCl concentrations (> 0.05 M) studied in the absence of divalent metals. Zn2+ causes higher order aggregation, but no such aggregates are seen with Mg2+ and Mn2+. In the presence of 1 mM manganese, the average lifetime decreased approximately 10%, from 8.14 ns to 7.38 ns, with a concomitant increase of average rotational correlational time (phi) from 24 ns to 28 ns. The accessibility of the positively charged quencher (Cs+) to tryptophan also decreases approximately 50%, indicating alteration of the tryptophan microenvironment. By contrast, Mg2+ causes minor changes in fluorescence properties. The hydrodynamic shape of the intact enzyme and its single-stranded (8 kDa) and double-stranded (31 kDa) DNA binding domains were further investigated by sedimentation velocity measurements. The value of S0(20),W for the intact enzyme is 2.97 S, and the calculated axial ratio is 5.0. In contrast to the 8 kDa domain, which has a less asymmetric shape with an axial ratio of 2.3, the 31 kDa domain shows an elongated structure with an axial ratio of 5.5. These data suggest that the axial ratio of the intact enzyme may be the result of marked bending of the molecule at the flexible hinge region between the two domains.
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PMID:Characterization of the tryptophan fluorescence and hydrodynamic properties of rat DNA polymerase beta. 796 32


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