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)

Adenosine 2',3'-riboepoxide 5'-triphosphate (epoxyATP) has been found to be a suicidal inactivator of DNA polymerase I from Escherichia coli by the following criteria. Inactivation is complete, is first order in enzyme activity, and shows saturation kinetics with an apparent KD of 30 +/- 10 micron for epoxy ATP. This KD is comparable to the KM of the substrate dATP. The t1/2 for inactivation is 1.3 min. Inactivation requires Mg2+ and the complementary template. The enzyme is protected by dATP but not by an excess of template. Gel filtration of the reaction mixture after inactivation with [3H]epoxy ATP results in the comigration of E. coli DNA polymerase I, the tritium-labeled inactivator, and the DNA template. The stoichiometry of binding approaches 1 mol of [3H]epoxy nucleotide per mol of inactivated enzyme. These results are consistent with the hypothesis that epoxy ATP initially serves as a substrate for the polymerase reaction, elongating the DNA chain by a nucleotidyl unit, and subsequently alkylates an essential base at the primer terminus binding site of the enzyme. Epoxy ATP also inactivates human and viral DNA polymerases but not E. coli RNA polymerase or rabbit muscle pyruvate kinase. Hence epoxy ATP may be a specific suicide reagent for DNA polymerases.
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PMID:Apparent suicidal inactivation of DNA polymerase by adenosine 2',3'-riboepoxide 5'-triphosphate. 34 91

The properties of the methyl-directed DNA (cytosine-5-)-methyltransferase (EC 2.1.1.37) suggest that it is the enzyme that maintains patterns of methylation in the human genome. Proposals for the enzyme's mechanism of action suggest that 5-methyldeoxycytidine is produced from deoxycytidine via a dihydrocytosine intermediate. We have used an oligodeoxynucleotide containing 5-fluorodeoxycytidine as a suicide substrate to capture the enzyme and the dihydrocytosine intermediate. Gel retardation experiments demonstrate the formation of the expected covalent complex between duplex DNA containing 5-fluorodeoxycytidine and the human enzyme. Formation of the complex was dependent upon the presence of the methyl donor S-adenosylmethionine, suggesting that it comprises an enzyme-linked 5-substituted dihydrocytosine moiety in DNA. Dihydrocytosine derivatives are extremely labile toward hydrolytic deamination in aqueous solution. Because C-to-T transition mutations are especially prevalent at CG sites in human DNA, we have used high-performance liquid chromatography to search for thymidine that might be generated by hydrolysis during the methyl transfer reaction. Despite the potential for deamination inherent in the formation of the intermediate, the methyltransferase did not produce detectable amounts of thymidine. The data suggest that the ability of the human methyltransferase to preserve genetic information when copying a methylation pattern (i.e., its fidelity) is comparable to the ability of a mammalian DNA polymerase to preserve genetic information when copying a DNA sequence. Thus the high frequency of C-to-T transitions at CG sites in human DNA does not appear to be due to the normal enzymatic maintenance of methylation patterns.
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PMID:Mechanism of human methyl-directed DNA methyltransferase and the fidelity of cytosine methylation. 158 13

These studies aimed to determine the expression and functional role of c-myb in erythroid progenitors with different cycling activities. In the first series of experiments the erythroid burst-forming unit (BFU-E) and colony-forming unit (CFU-E) populations from adult peripheral blood (PB), bone marrow (BM), and embryonic-fetal liver (FL) were treated with either c-myb antisense oligomers or 3H-thymidine (3H-TdR). A direct correlation was always observed between the inhibitory effect of anti-myb oligomers and the level of cycling activity. Thus, the inhibitory effect of antisense c-myb on the number of BFU-E colonies was 28.3% +/- 15.8% in PB, 53.4% +/- 9.3% in BM, and 68.2% +/- 24.5% in FL. Both adult and embryonic CFU-E were markedly inhibited (73.2% +/- 10.4% and 74.2% +/- 12.7%). Using highly purified PB progenitors, we observed a similar pattern, although with slightly lower inhibitory effects. In the 3H-TdR suicide assay the killing index of BFU-E was 8.9% +/- 4.2% in PB, 29.4% +/- 6.5% in BM, and 40.1% +/- 9.6% in FL. The values for adult and embryonic CFU-E were 55.7% +/- 7.9% and 60.98% +/- 6.6%, respectively. We then investigated the kinetics of c-myb mRNA level during the erythroid differentiation of highly purified adult PB and FL BFU-E, as evaluated in liquid-phase culture by reverse transcription-polymerase chain reaction. Adult erythroid precursors showed a gradual increase of c-myb mRNA from day 4 through day 8 of culture and a sharp decrease at later times, whereas the expression of c-myb mRNA and protein in differentiation embryonic precursors peaked 2 days earlier. In both cases, c-myb mRNA level peaked at the CFU-E stage of differentiation. Finally, highly purified adult PB BFU-E were stimulated into cycling by a 3-day treatment with interleukin-3 in liquid phase: both the sensitivity to c-myb antisense oligomers and the 3H-TdR suicide index showed a gradual, strictly parallel increase. Under the same experimental conditions a progressive increase of the mRNA level of DNA polymerase alpha was observed. These observations suggest that in early erythroid differentiation c-myb activation is associated with the progression of progenitors into the S phase of the cell cycle, as well as to the synthesis of DNA polymerase alpha.
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PMID:Antisense myb inhibition of purified erythroid progenitors in development and differentiation is linked to cycling activity and expression of DNA polymerase alpha. 170 31

Metabolic activation of cyclophosphamide (CP) by microsomal mixed function hydroxylases yields 4-hydroxycyclophosphamide and aldophosphamide defined as activated CP. Activated CP shows relatively high cancerotoxic selectivity in vivo and cytotoxic specificity in vitro and can be trapped rapidly by reversible reaction of hemiaminal group of the oxazaphosphorine ring with protein thiols to form protein bound activated CP (protein-S-CP). Protein-S-CP stores activated CP in a highly stable form. From pharmacokinetics of activated CP in mice after the injection of cyclophosphamide, it was calculated that about 17% of the CP dose given was stored in a pool of protein bound activated CP lasting for several days. From therapy studies with 4-(S-ethanol)-sulfido-CP in combination with excess of cysteine, it was concluded that the protein-S-CP pool may be that form of activated CP which is mainly responsible for the specific cytotoxic effects in the tumor cells. On the other hand free unbound 4-OH-CP was shown to contribute mainly to overall toxicity. No spontaneous toxicogenation of activated CP was noted under in vivo conditions. 3'-5' Exonucleases were found to hydrolyze 4-OH-CP, yielding phosphoramide mustard and acrolein as split products. Because of the low affinity of 4-OH-CP for plain 3'-5' exonucleases, it seems however unlikely that these enzymes play a major role in the antitumor effect of CP in vivo. 3'-5' Exonucleases associated to DNA polymerase like in DNA polymerase delta from rabbit bone marrow or in DNA polymerase I from E. coli are more likely candidates for 4-OH-CP toxicogenation because of the much higher specific activity with 4-OH-CP as substrate. In experiments with DNA polymerase I from E. coli, 4-OH-CP was shown to inhibit DNA polymerase activity after toxicogenation by the 3'-5' exonuclease subsite of the enzyme. This suggests an enzyme mechanism based suicide inactivation of the DNA polymerase. Because of the close spatial cooperation of the DNA polymerase and 3'-5' exonuclease subsites with primer/template a site-specific alkylation of DNA is also postulated. Thus we raised the hypothesis that cytotoxic specificity of activated CP is based on the interaction of protein-S-CP (protein bound activated CP) with DNA polymerase/3'-5' exonuclease as the target. In a P 815 mouse mast-cell tumor we determined by means of 5' AMP agarose affinity chromatography two/third of total DNA polymerase to be associated with 3'-5' exonuclease.
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PMID:The enzymatic basis of cyclophosphamide specificity. 302 54

Thiazolidinyl- and perhydrothiazinyl-ethyl-N-mustard-phosphamide esters were designed to act as highly specific suicide inactivators of DNA polymerase alpha holoenzymes. Acute and subacute toxicity of these drugs in mice was very small. By daily i.p. injection, on day 0-4 mice were cured of P 388 lymphatic leukaemia with no depression of blood leucocytes. The findings suggest that suicide inactivators of DNA polymerase alpha holoenzyme may be promising drugs for low toxicity cancer chemotherapy.
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PMID:Low toxicity cancer chemotherapy by suicide inactivation of DNA polymerase alpha holoenzyme: first results with new thiazolidinyl- and perhydrothiazinyl-ethyl-N-mustard-phosphamide esters. 338 44

The triphosphate form of 9-[(2-hydroxyethoxy)-methyl]guanine (acyclovir), ACVTP, inactivates the herpes simplex virus type 1 DNA polymerase. ACVTP does not innately inactivate resting polymerase, but becomes an inactivator only while being processed as an alternative substrate. Pseudo first-order rates of inactivation were measured at varying concentrations of ACVTP and fixed concentrations of the natural substrate, deoxyguanosine triphosphate. These studies indicated that a reversible enzyme-ACVTP (Michaelis-type) complex is formed at the active site prior to inactivation. The formation of this complex was competitively retarded by deoxyguanosine triphosphate. An apparent dissociation constant (KD) of 3.6 +/- 0.2 (S.D.) nM was determined for ACVTP from this reversible complex. A second method for the estimation of the KD which used the extrapolated initial velocities produced a value of 5.9 +/- 0.4 (S.D.) nM. The rate of conversion of the reversible complex to the inactivated complex, at saturating ACVTP, was calculated to be 0.24 min-1. No reactivation of enzyme activity was detected following isolation of the inactivated complex by rapid desalting on Sephadex G-25. Under these conditions, an overall reactivation rate of 1.5 X 10(-5) min-1 could have been easily detected. Therefore, the overall inhibition constant must have been less than 3 pM. In contrast, when host DNA polymerase alpha was incubated with 14 microM ACVTP, only 60% inhibition of enzyme activity was observed, but inactivation was not detected. These data indicate that ACVTP functions as a suicide inactivator of the herpes simplex virus type 1 DNA polymerase, and is only a weak reversible inhibitor of DNA polymerase alpha.
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PMID:Acyclovir triphosphate is a suicide inactivator of the herpes simplex virus DNA polymerase. 608 40

Studying the activity of T7 DNA polymerase (Sequenase) on open circular DNAs, we observed virtually complete termination within potential triplex-forming sequences. Mutations destroying the triplex potential of the sequences prevented termination, while compensatory mutations restoring triplex potential restored it. We hypothesize that strand displacement during DNA polymerization of double-helical templates brings three DNA strands (duplex DNA downstream of the polymerase plus a displaced overhang) into close proximity, provoking triplex formation, which in turn prevents further DNA synthesis. Supporting this idea, we found that Sequenase is unable to propagate through short triple-helical stretches within single-stranded DNA templates. Thus, DNA polymerase, by inducing triplex formation at specific sequences in front of the replication fork, causes self-termination. Possible biological implications of such 'conformational suicide' are discussed. Our data also provide a novel way to target DNA polymerases at specific sequences using triplex-forming oligonucleotides.
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PMID:Suicidal nucleotide sequences for DNA polymerization. 826 40

Most antiherpes therapies exploit the large substrate acceptance of herpes simplex virus type 1 thymidine kinase (TK(HSV1)) relative to the human isoenzyme. The enzyme selectively phosphorylates nucleoside analogs that can either inhibit viral DNA polymerase or cause toxic effects when incorporated into viral DNA. To relate structural properties of TK(HSV1) ligands to their chemical reactivity we have carried out ab initio quantum chemistry calculations within the density functional theory framework in combination with biochemical studies. Calculations have focused on a set of ligands carrying a representative set of the large spectrum of sugar-mimicking moieties and for which structural information of the TK(HSV1)-ligand complex is available. The k(cat) values of these ligands have been measured under the same experimental conditions using an UV spectrophotometric assay. The calculations point to the crucial role of electric dipole moment of ligands and its interaction with the negatively charged residue Glu(225). A striking correlation is found between the energetics associated with this interaction and the k(cat) values measured under homogeneous conditions. This finding uncovers a fundamental aspect of the mechanism governing substrate diversity and catalytic turnover and thus represents a significant step toward the rational design of novel and powerful prodrugs for antiviral and TK(HSV1)-linked suicide gene therapies.
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PMID:The rational of catalytic activity of herpes simplex virus thymidine kinase. a combined biochemical and quantum chemical study. 1126 92

Oxidized abasic residues in DNA constitute a major class of radiation and oxidative damage. Free radical attack on the nucleotidyl C-1' carbon yields 2-deoxyribonolactone (dL) as a significant lesion. Although dL residues are efficiently incised by the main human abasic endonuclease enzyme Ape1, we show here that subsequent excision by human DNA polymerase beta is impaired at dL compared with unmodified abasic sites. This inhibition is accompanied by accumulation of a protein-DNA cross-link not observed in reactions of polymerase beta with unmodified abasic sites, although a similar form can be trapped by reduction with sodium borohydride. The formation of the stably cross-linked species with dL depends on the polymerase lysine 72 residue, which forms a Schiff base with the C-1 aldehyde during excision of an unmodified abasic site. In the case of a dL residue, attack on the lactone C-1 by lysine 72 proceeds more slowly and evidently produces an amide linkage, which resists further processing. Consequently dL residues may not be readily repaired by "short-patch" base excision repair but instead function as suicide substrates in the formation of protein-DNA cross-links that may require alternative modes of repair.
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PMID:Covalent trapping of human DNA polymerase beta by the oxidative DNA lesion 2-deoxyribonolactone. 1180 79

Generation of adenovirus-based vectors through homologous recombination within Escherichia coli cells is one of the most efficient strategies. A common challenge associated with this method is the formation of colonies containing self-ligated shuttle plasmid. To improve homologous recombination, a new pAdEasy-1-bearing competent cell line was constructed so that it no longer requires co-transformation with two plasmids and can generate more recombinant colonies (ninefold). New and efficient approaches were also tested to block shuttle plasmid self-ligation by a combined treatment of the plasmid with Taq DNA polymerase and calf intestine phosphatase (CIP) or blocking the formation of self-ligated plasmid-containing colonies by subcloning a suicide gene, ccdB, into the plasmid construct. Present experimental data show that these modifications are effective in eliminating self-ligated plasmid-containing colony background and offer greater simplicity, faster experimental progress, and higher efficiency in performing homologous recombination within E. coli cells, which could facilitate the production of high-titer infectious viral particles.
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PMID:Effective modifications for improved homologous recombination and high-efficiency generation of recombinant adenovirus-based vectors. 1875 19


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