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)

The dnaZX gene of Escherichia coli directs the synthesis of two proteins, DnaZ and DnaX. These products are confirmed as the gamma and tau subunits of DNA polymerase III because antibody to a synthetic peptide present in both the DnaZ and DnaX proteins reacts also with the gamma and tau subunits of holoenzyme. To characterize biochemically the tau subunit, for which there has been no activity assay, the dnaZX gene was fused to the beta-galactosidase gene to encode a fusion product in which the 20 C-terminal amino acids of the DnaX protein (tau) were replaced by beta-galactosidase lacking only 7 N-terminal amino acids. The 185-kDa fusion protein, which retained beta-galactosidase activity, was overproduced to the level of about 5% of the soluble cellular protein by placing the gene fusion under control of the tac promoter and Shine-Dalgarno sequence. The fusion protein was isolated in one step by affinity chromatography on p-aminobenzyl 1-thio-beta-D-galactopyranoside-agarose. The purified fusion protein also had ATPase (and dATPase) activity that was dependent on single-stranded DNA. This activity copurified with the beta-galactosidase activity not only through the affinity column but also through a subsequent gel filtration. We conclude that the DnaX protein function involves binding to single-stranded DNA and hydrolysis of ATP or dATP, in addition to binding to other DNA polymerase III holoenzyme components, increasing the processivity of the core enzyme, and serving as a substrate for the production of the gamma subunit.
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PMID:Escherichia coli DnaX product, the tau subunit of DNA polymerase III, is a multifunctional protein with single-stranded DNA-dependent ATPase activity. 303 60

A.A mismatch errors occurring during poly(dA) replication with the Klenow fragment of E. coli DNA polymerase I have been quantified. The A/T ratio measured for chains extended by 1-25 nucleotides decreases by a factor of at least 15 from beginning to end. The deduced true error rate may decrease by a factor of 2.5 at each successive nucleotide addition. When ddATP is used instead of dATP, the ddA/T ratio indicates little variation of the misincorporation probability with position. Thus, the accuracy improvement in the first case is due to a warm-up of the proofreading function.
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PMID:Variations with position of replication errors due to exonuclease warm-up. 304 Apr 69

Recently, 2-halogenated deoxyadenosine analogs (F, Cl, and Br) have been shown to have antitumor activity. These analogs are phosphorylated by cells and are believed to exert their cytotoxic action at the nucleoside triphosphate level. In this work the interaction of these nucleoside triphosphate analogs with potential targets, such as DNA polymerase alpha, beta, and gamma, DNA primase, and ribonucleotide reductase was examined in detail. All of these compounds competitively inhibited the incorporation of dAMP into DNA by DNA polymerase alpha, beta, or gamma. F-dATP was able to completely substitute for dATP using DNA polymerase alpha and gamma, but not with DNA polymerase beta. Cl-dATP and Br-dATP substituted poorly for dATP using DNA polymerase alpha and beta. Extension of a 32P-labeled primer by DNA polymerase alpha, beta, or gamma on a single-stranded M13 template showed that these compounds were incorporated into the 3' end of the growing DNA chain and that elongation beyond the incorporated analogs was significantly retarded for Cl-dATP and Br-dATP using either DNA polymerase alpha or beta. DNA primase using poly(dC) as template was inhibited by these compounds at a concentration 4 to 5 times greater than that required for 2-F-araATP. The 2-halogenated dATP analogs were potent inhibitors of ADP reduction by ribonucleotide reductase. In conclusion, the cytotoxic action of 2-Cl-deoxyadenosine and 2-Br-deoxyadenosine may partially be mediated through the mechanism of "self-potentiation," by depression of the deoxynucleoside triphosphate pools due to inhibition of ribonucleotide reductase, which would facilitate their incorporation into DNA and result in the inhibition of DNA synthesis.
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PMID:Interaction of 2-halogenated dATP analogs (F, Cl, and Br) with human DNA polymerases, DNA primase, and ribonucleotide reductase. 305 Apr 47

The phi 29 protein p6 stimulates the formation of the protein p3-dAMP initiation complex when added to a minimal system containing the terminal protein p3, the phi 29 DNA polymerase p2 and phi 29 DNA-protein p3 complex, by decreasing about 5 fold the Km value for dATP. In addition, protein p6 stimulates elongation of the p3-dAMP initiation complex. Whereas the effect of protein p6 on initiation is similar with protein p3-containing fragments from the right or left phi 29 DNA ends, the stimulation of elongation is higher with the right than with the left phi 29 DNA terminal fragment, suggesting DNA sequence specificity. The stimulation by protein p6 of the initiation and elongation steps of phi 29 DNA replication does not require the presence of the parental protein p3 at the phi 29 DNA ends. No effect of protein p6 was obtained on the elongation of the template-primer poly(dT)-(dA) 12-18 by the phi 29 DNA polymerase.
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PMID:Replication of phage phi 29 DNA in vitro: role of the viral protein p6 in initiation and elongation. 308 45

The drugs aphidicolin and the nucleotide analogs butylanilino dATP, butylphenyl dGTP, and butylphenyl rGTP inhibited the protein-primed replication of phi 29 DNA-protein p3 in the presence of purified terminal protein p3 and phi 29 DNA polymerase p2. The effect of aphidicolin was mainly on the polymerization reaction by decreasing the rate of elongation. The nucleotide analogs inhibited both the formation of the p3-dAMP initiation complex and its further elongation, the latter being also due to a decrease in the elongation rate. When assayed with the phi 29 DNA polymerase as the only protein, all the drugs inhibited polymerization on activated DNA as well as the 3'----5' exonuclease activity of the polymerase, indicating that the target of the drugs is the phi 29 DNA polymerase itself.
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PMID:Effect of aphidicolin and nucleotide analogs on the phage phi 29 DNA polymerase. 309 Jul 78

The authors' approach to the design of DNA polymerase-specific inhibitors, an approach based on the mechanism of action of 6-(p-hydroxyphenylazo)uracil, has been to disguise nucleic acid bases to mimic the purine substrates dGTP and dATP. Specifically, the strategy has been to synthesize bases with substituents that endow them with the capacity: to seek and react with unique features of the active site of a polymerase; and to form H bonds with complementary template pyrimidines apposing the active site. This strategy has yielded a series of novel, enzyme-specific dATP and dGTP analogues which are non-polymerizable and which inhibit their target polymerase by sequestering it to a complementary pyrimidine residue in primer:template. The work has involved primarily two replication-specific polymerases, B. subtilis DNA polymerase III (pol III) and mammalian DNA polymerase alpha (pol alpha). The initial design exploited the pyrimidine nucleus and produced inhibitors with Ki values in the micromolar range. Principles established with the pyrimidine derivatives have led to the development of bona fide purine nucleotide analogues which act as DNA polymerase inhibitors of high selectivity and unprecedented potency. For example, BuPdGTP, the 2'-deoxyribonucleoside 5'-triphosphate of N2-(p-n-butylphenyl)guanine (BuPG), lacks discernible activity against mammalian polymerases beta and gamma, whereas it inhibits mammalian pol alpha with a Ki of less than 10 nanomolar. Currently, the authors are exploiting BuPdGTP, BuPdGDP, and similar butylanilino derivatives of dATP to probe the active site of pol alpha and to develop other N2-substituted analogues which can bind selectively to the substrate sites of other important polymerases and nucleotide binding proteins.
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PMID:Rational design of substrate analogues targeted to selectively inhibit replication-specific DNA polymerases. 309 44

The mode of action of aphidicolin on DNA synthesis catalysed by the DNA polymerase of Methanococcus vannielii is competitive for dCTP, noncompetitive for dATP, dGTP and dTTP and uncompetitive for activated DNA. The kinetic data are accounted for by a mechanism in which dCTP and aphidicolin compete for the dCTP-specific binding site on the DNA polymerase. The dissociation constant for the aphidicolin--DNA-polymerase complex is 0.04-0.07 microM. Similar modes of inhibition of DNA synthesis exist for DNA polymerase alpha of higher eucaryotes but not for eubacteria or viruses and suggests a close functional relationship between the DNA polymerase of eucaryotes and of the archaebacterium M. vannielii.
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PMID:Mode of inhibition of the DNA polymerase of Methanococcus vannielii by aphidicolin. 310 39

From among a series of stable, aphidicolin-resistant mutant strains of mouse teratocarcinoma, derived from a multipotent parental line (PSA-1-80), three were selected for further study on the basis of their comparatively high degrees of resistance and elevated frequencies of spontaneous forward mutation to 6-thioguanine and ouabain resistance. Fluctuation tests confirmed that they were mutator strains. Since each of the three mutants was isolated after multiple rounds of selection, and since a variety of biochemical abnormalities were observed, it is likely that a number of mechanisms, probably consisting of overlapping subsets, determine the phenotypes. Abnormalities in the metabolism of the nucleotide substrates for polymerization are likely to be of major importance in mutants designated Aph-2 and Aph-3, as there were marked alterations in the dCTP and dATP pool sizes. The specific activity of DNA polymerase alpha was also increased. For the case of Aph-3, which exhibited the greatest (400-fold) increase in resistance to aphidicolin, a mutation in the structural gene for DNA polymerase alpha may be an additional important component, since in vitro assays revealed that the isolated enzyme was resistant to aphidicolin. For the case of Aph-1 however, only minor alterations in dNTP pools were observed, and there was no increase in the specific activity of DNA polymerase alpha or in the aphidicolin resistance of the isolated DNA polymerase alpha, suggesting yet another mechanism(s) underlying the aphidicolin resistance/mutator phenotype. All three mutants formed subcutaneous tumors in syngeneic mice; both Aph-1 and Aph-2 were multipotent; whereas Aph-3 was nullipotent.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Aphidicolin-resistant mutator strains of mouse teratocarcinoma. 311 23

Sinefungin, a natural nucleoside isolated from cultures of Streptomyces incarnatus and S. griseolus, is structurally related to S-adenosylhomocysteine and S-adenosylmethionine. Sinefungin has been shown to inhibit the development of various fungi and viruses, but its major attraction to date resides in its potent antiparasitic activity. This compound has been reported to display antiparasitic activity against malarial, trypanosomal, and leishmanial species. Very little is known about the antiparasitic mode of action of sinefungin. We found that S-adenosylmethionine was capable of reversing the inhibitory growth effects of sinefungin in Leishmania mexicana and that dATP was capable of reversing inhibitory effects of the drug on DNA polymerase activity when pyrophosphate release was measured. However, when incorporation of [3H]dTTP was used to measure DNA polymerase activity, no inhibition could be observed. Inhibition of DNA polymerase activity by sinefungin occurred only during the initial stages of purification of this enzyme, and inhibition by aphidicolin, a known DNA polymerase inhibitor, paralleled the inhibition by sinefungin. Neither sinefungin nor aphidicolin inhibited partially purified DNA polymerase. S-Adenosylmethionine synthetase was partially purified, and sinefungin, at levels active in vitro, had no significant effect. Sinefungin was significantly suppressive against both L. donovani and L. braziliensis panamensis infections in hamsters when compared with meglumine antimonate (Glucantime).
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PMID:Molecular target of the antileishmanial action of sinefungin. 312 33

The Bacillus subtilis phage luminal diameter 29 DNA polymerase, involved in protein-primed viral DNA replication, was inhibited by phosphonoacetic acid (PAA), a known inhibitor of alpha-like DNA polymerases, by decreasing the rate of elongation. Three highly conserved regions of amino acid homology, found in several viral alpha-like DNA polymerases and in the luminal diameter 29 DNA polymerase, one of them proposed to be the PAA binding site, were also found in the T4 DNA polymerase. This prokaryotic enzyme was highly sensitive to the drugs aphidicolin and the nucleotide analogues butylanilino dATP (BuAdATP) and butylphenyl dGTP (BuPdGTP), known to be specific inhibitors of eukaryotic alpha-like DNA polymerases. Two potential DNA polymerases from the linear plasmid pGKL1 from yeast and the S1 mitochondrial DNA from maize have been identified, based on the fact that they contain the three conserved regions of amino acid homology. Comparison of DNA polymerases from prokaryotic and eukaryotic origin showed extensive amino acid homology in addition to highly conserved domains. These findings reflect evolutionary relationships between hypothetically unrelated DNA polymerases.
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PMID:Structural and functional relationships between prokaryotic and eukaryotic DNA polymerases. 312 4


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