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)

The antiherpetic agent 9-[(2,3-dihydroxy-1-propoxy)methyl]guanine (iNDG) is phosphorylated by HSV1 thymidine kinase, and its phosphorylated products inhibit DNA polymerase activity. iNDG exists in two enantiomeric forms, each with a primary and a secondary hydroxyl; thus, a number of possibilities for preferential phosphorylation exist, which were explored in this study. HSV1 thymidine kinase phosphorylates the primary hydroxyl of both the R and the S isomers of iNDG. This was established by comparison with analogues in which either the primary or the secondary hydroxyl was replaced by fluorine or hydrogen and also by a study of the NMR spectrum of the monophosphate. GMP kinase phosphorylates the R and the S monophosphates to the respective diphosphates. Further phosphorylation, however, is much more efficient with the S than with the R isomer. Furthermore, (S)-iNDG triphosphate is a more potent inhibitor of HSV1 DNA polymerase than (R)-iNDG triphosphate. These differences in the biochemical specificities of the two isomers account for the observed higher antiviral potency of (S)-iNDG as compared to that of (R)-iNDG.
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PMID:Enzymatic phosphorylation of the antiherpetic agent 9-[(2,3-dihydroxy-1-propoxy)methyl]guanine. 300 16

The affinities of oligothymidylates and of some analogs for the template site, of a set of oligodeoxyribo- and oligoribonucleotides for the primer site, and of dNTPs and some analogs for the substrate sites of DNA polymerase I Klenow fragment and of human placenta DNA polymerase alpha were measured using them either as competitors of affinity modification or as substrates. The data obtained enable us to hypothesize that the Me2+-dependent electrostatic contact and hydrogen bond of a single internucleotide phosphate and the hydrophobic interactions of the other nucleotide units determine the formation of oligonucleotide-template site complexes. Interaction of the primer's 3'-terminal hydroxy group and of the negatively charged adjacent phosphate with the enzyme, and Watson-Crick base pairing with the template are of crucial importance for the formation of the ternary enzyme-template-primer complex. dNTP and dNMP imidazolides inactivate enzymes via an affinity modification mechanism only in the presence of the template-primer complex. dNTP affinities exceed those of dNDPs and dNMPs, the enhancement being most significant for the substrate that is complementary to the template, thus suggesting the participation of the gamma-phosphate of dNTP in the substrate selection step.
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PMID:Protein-nucleic acid interaction in reactions catalyzed with DNA polymerases. 313 84

Thioredoxin is a small (Mr 12,000) ubiquitous redox protein with the conserved active site structure: -Trp-Cys-Gly-Pro-Cys-. The oxidized form (Trx-S2) contains a disulfide bridge which is reduced by NADPH and thioredoxin reductase; the reduced form [Trx(SH)2] is a powerful protein disulfide oxidoreductase. Thioredoxins have been characterized in a wide variety of prokaryotic cells, and generally show about 50% amino acid homology to Escherichia coli thioredoxin with a known three-dimensional structure. In vitro Trx-(SH)2 serves as a hydrogen donor for ribonucleotide reductase, an essential enzyme in DNA synthesis, and for enzymes reducing sulfate or methionine sulfoxide. E. coli Trx-(SH)2 is essential for phage T7 DNA replication as a subunit of T7 DNA polymerase and also for assembly of the filamentous phages f1 and M13 perhaps through its localization at the cellular plasma membrane. Some photosynthetic organisms reduce Trx-S2 by light and ferredoxin; Trx-(SH)2 is used as a disulfide reductase to regulate the activity of enzymes by thiol redox control. Thioredoxin-negative mutants (trxA) of E. coli are viable making the precise cellular physiological functions of thioredoxin unknown. Another small E. coli protein, glutaredoxin, enables GSH to be hydrogen donor for ribonucleotide reductase or PAPS reductase. Further experiments with molecular genetic techniques are required to define the relative roles of the thioredoxin and glutaredoxin systems in intracellular redox reactions.
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PMID:Thioredoxin and related proteins in procaryotes. 315 90

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

O4-Methyl-, O4-ethyl-, and O4-isopropylthymidine 5'-triphosphates, which can be formed by N-nitroso carcinogens, were tested for their ability to substitute for thymidine 5'-triphosphate (dTTP) in synthesis catalyzed by Escherichia coli DNA polymerase I (Pol I) by using activated DNA or synthetic polymers as templates. All could substitute for dTTP for short periods, the rate and extent decreasing with the size of the alkyl group. Because the structure of O4-alkylthymidine does not permit normal hydrogen bond formation with deoxyadenosine, it was inferred that eventual formation of a poor or frayed primer end was responsible for termination of synthesis. Synthesis of polymers at temperatures ranging from 0 to 40 degrees C showed that the extent of incorporation using the O4-alkyl-dTTPs was favored, relative to dTTP, when the terminal helical structure was stabilized by low temperatures. Kmapp values were determined for each O4-alkyldeoxynucleoside 5'-triphosphate. These values were 0.7 microM for dTTP, 5 microM for methyl-dTTP, 11 microM for ethyl-dTTP, and 33 microM for isopropyl-dTTP. O4-Alkyl-dTTPs were tested for their ability to inhibit or compete with dTTP incorporation and found to have a minimal effect, even when present at high concentration. These experiments indicated that Pol I can incorporate deoxynucleotides with O4-alkyl substituents into an ordered DNA structure. A postulated base-pairing scheme with deoxyadenosine is described.
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PMID:O4-Methyl-, O4-ethyl-, and O4-isopropylthymidine 5'-triphosphates as analogues of thymidine 5'-triphosphate: kinetics of incorporation by Escherichia coli DNA polymerase I. 351 16

The repair response of Escherichia coli to hydrogen peroxide-induced DNA damage was investigated in intact and toluene-treated cells. Cellular DNA was cleaved after treatment by hydrogen peroxide as analyzed by alkaline sucrose sedimentation. The incision step did not require ATP or magnesium and was not inhibited by N-ethylmaleimide (NEM). An ATP-independent, magnesium-dependent incorporation of nucleotides was seen after the exposure of cells to hydrogen peroxide. This DNA repair synthesis was not inhibited by the addition of NEM or dithiothreitol. In dnaB(Ts) strain CRT266, which is thermolabile for DNA replication, normal levels of DNA synthesis were found at the restrictive temperature (43 degrees C), showing that DNA replication was not necessary for this DNA synthesis. Density gradient analysis also indicated that hydrogen peroxide inhibited DNA replication and stimulated repair synthesis. The subsequent reformation step required magnesium, did not require ATP, and was not inhibited by NEM, in agreement with the synthesis requirements. This suggests that DNA polymerase I was involved in the repair step. Furthermore, a strain defective in DNA polymerase I was unable to reform its DNA after peroxide treatment. Chemical cleavage of the DNA was shown by incision of supercoiled DNA with hydrogen peroxide in the presence of a low concentration of ferric chloride. These findings suggest that hydrogen peroxide directly incises DNA, causing damage which is repaired by an incision repair pathway that requires DNA polymerase I.
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PMID:Repair response of Escherichia coli to hydrogen peroxide DNA damage. 353 65

The mechanism of binding and elongation of the oligothymidylate primers in the systems of the DNA polymerase alpha from human placenta and DNA polymerase I from E. coli with the poly(dA) as a template was investigated. Both dTMP and dTTP were shown to be the minimal primers of DNA polymerase alpha, the affinity and V increasing 1.8- and 1.4-fold respectively upon lengthening the primer by each unit from dTMP to d(Tp)9T. Further elongation is accompanied by 1.3-fold affinity enhancement and a decrease in V. For the E. coli enzyme, a similar dependence of affinity of primer d(Tp)4T-d(Tp)14T was observed with the inflexion point corresponding to d(Tp)8T. The individual diastereomers of oligothymidylate ethyl esters (with p' and p'' corresponding to enantiomeric configuration) such as d[Tp'(Et)Tp]3Tp'(Et)T, d[Tp''(Et)Tp]3Tp''(Et)T, d(Tp)8Tp'(Et)T, d(Tp)8Tp''(Et)T, d(Tp)8Tp'(Et)TpT, d(Tp)8 X X Tp''(Et)TpT and completely esterified analogues d[Tp(Et)]7T, d[Tp(Et)]14T were shown to initiate the poly (dA)-dependent polymerization catalyzed by both enzymes. A sum of the obtained results provided the basis for a number of conjectures on the mode of primer and template binding to the enzyme, possible role of their preformed complex, as well as electrostatic interactions and hydrogen bonding.
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PMID:[Prokaryotic and eukaryotic DNA-polymerase. I. The role of internucleotide phosphate groups in the binding of a primer with the enzyme]. 355 63

The affinity of different ligands (phosphate, nucleoside monophosphates, oligonucleotides) to the template binding site of DNA polymerase alpha from human placenta was estimated. To this goal, dependences of rate of the enzyme inactivation by the affinity reagent d(pT)2pC[Pt2+(NH3)2OH](pT)7 on the concentration of these ligands as competitive inhibitors were determined. Minimal ligands capable to bind with the template site of DNA polymerase alpha were shown to be triethylphosphate (Kd 600 microM) and phosphate (Kd 53 microM). Ligand affinity increases by the factor 1.71 per added monomer unit from phosphate to d(pT) and then for oligothymidylates d(Tp)nT (n 1 to 14). The partial ethylation of phosphodiester groups does not change the efficiency of the oligothymidylate binding with the enzyme. However, the complete ethylation of these groups lowers affinity of the oligothymidylates to the enzyme by 7-9 times. The decrease is comparable with the change of Pt2+-decathymidylate affinity to the enzyme caused by Mn2+-ions. The data obtained led to suggestion that an electrostatic contact (most likely, Me2+-dependent) of phosphodiester group with the enzyme takes place. The type of contact is confirmed by Gibbs' energy change 1.1-1.4 kcal/mole. Formation of a hydrogen bond with the oxygen atom of P = O group of the same phosphate is also assumed (delta G =--4.4 . . .--4.5 kcal/mole). The other internucleotide phosphates and all bases of oligonucleotides form neither hydrogen bonds nor electrostatic contacts with the template binding site. Gibbs' energy changes by 0.32 kcal/mole when the template is lengthened by one unit. We suppose that this value characterizes the energy gain in the transition of oligonucleotide template from aquous medium to the hydrophobic environement of the enzyme active site. Comparison of Km values of oligothymidylates and their partially or completely ethylated analogues as templates in the reaction of DNA polymerization catalysed by DNA polymerase alpha from human placenta and Klenow's fragment of E. coli DNA polymerase I suggests a similar mechanism of template recognition by both enzymes.
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PMID:[Eukaryotic and prokaryotic DNA-polymerase. II. The role of internucleotide phosphate groups of a template in its binding with the enzyme]. 355 64

Affinity labelling of human placenta DNA polymerase alpha (EC 2.7.7.7) with the reactive oligodeoxyribonucleotide d(pT)2pC[Pt2+(NH3)2OH](pT)7 was used for quantitative analysis of enzyme interaction with oligodeoxyribonucleotides as templates. Dissociation constants and Gibb's energy values for different oligothymidylates d(pT)nT where n = 1-14 have been evaluated by competitive experiments of these ligands with Pt2+ reagent. The data obtained prove the formation of one Me2+-dependent electrostatic contact and a hydrogen bond between the enzyme and one phosphate of these templates. One may suppose that the hydrophobic interaction of any other monomeric link of oligodeoxyribonucleotides with the enzyme template site takes place.
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PMID:Role of nucleoside components and internucleotide phosphate groups of oligodeoxyribonucleotide template in its binding to human DNA polymerase alpha. 358 74

The cytostatic potential of twenty antibiotic agroclavines has been examined in the L5178y mouse lymphoma cell system. Twelve of these compounds are described for the first time. It is shown that the substituent at N-1 of agroclavine is very important whereas the substituent at N-6 is of less influence if it is not hydrogen. Incorporation studies in the presence of 1-propylagroclavine suggest that DNA synthesis in the lymphoma cells is inhibited. The effect on the corresponding [3H]thymidine incorporation in murine spleen lymphocytes is comparably low. Neither a significant change of mRNA efflux nor of DNA polymerase alpha and beta activities was caused. The mechanism of action seems to be a fundamentally new one for ergoline compounds as interactions with alpha-adrenoceptors, dopamine and 5-hydroxytryptamine receptors are not involved.
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PMID:Clavines as antitumor agents, 3: Cytostatic activity and structure/activity relationships of 1-alkyl agroclavines and 6-alkyl 6-noragroclavines. 373 28


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