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

---

Query: EC:2.7.7.7 (DNA polymerase)
17,007 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Escherichia coli contains three DNA polymerases that differ in their size, ability to interact with accessory proteins and biological function. Monomeric DNA polymerase I (Pol I) has a relative molecular mass (Mr) of 103,000 (103K) and is involved primarily in the repair of damaged DNA and the processing of Okazaki fragments; polymerase II is of Mr 120K, and polymerase III has a Mr of 140K, is responsible for the replication of the DNA chromosome and is just one of several proteins that are required for replication. DNA polymerases from bacteriophage as well as those of eukaryotic viral and cellular origin also differ with respect to their size and the number of associated proteins that are required for them to function in replication. However, the template-directed copying of DNA is identical in all cases. The crystal structure of the large proteolytic fragment of Pol I shows that it consists of two domains, the larger of which contains a deep crevice whose dimensions are such that it can bind duplex DNA. The T7 polymerase consists of two subunits, the 80K gene 5 protein and the host-encoded 12K thioredoxin of E. coli. We show here that there is an amino acid sequence homology between at least eight polypeptide segments that form the large cleft in the Klenow fragment and polypeptides in T7 DNA polymerase gene 5 protein, suggesting that this domain evolved from a common precursor. The parts of the Pol I and T7 DNA polymerase molecules that bind the DNA substrate appear to share common structural features, and these features may be shared by all of these varied DNA polymerases.
...
PMID:Domain of E. coli DNA polymerase I showing sequence homology to T7 DNA polymerase. 388 96

Bacteriophage T7-induced DNA polymerase is composed of a 1:1 complex of phage-induced gene 5 protein and Escherichia coli thioredoxin. Preparation of active subunits in the absence of sulfhydryl reagents indicates the reduced form of thioredoxin is sufficient for formation of the active holoenzyme. The oxidized form of thioredoxin, thioredoxin modified at one active site sulfhydryl by iodoacetate or methyl iodide, or thioredoxin modified at both active site sulfhydryls by N-ethylmaleimide, are all inactive, being defective in complex formation with gene 5 protein. Thioredoxin sulfhydryl groups present in native T7 DNA polymerase do not appear to be involved in an intersubunit disulfide bond; one and probably both sulfhydryls are available in the native holoenzyme for modification by N-ethylmaleimide. Furthermore, DNA substrates alter the reactivity of thioredoxin cysteines within the holoenzyme with respect to this reagent. Substrates for the single strand exonuclease enhance the reactivity of thioredoxin sulfhydryl groups while those for the polymerase or double strand exonuclease functions afford protection. It, therefore, seems likely that thioredoxin sulfhydryl groups are present in the reduced state within the native polymerase.
...
PMID:T7-induced DNA polymerase. Requirement for thioredoxin sulfhydryl groups. 634 83

Phage T7 DNA polymerase purified to homogeneity by an antithioredoxin immunoadsorbent technique was resolved into its active subunits the gene 5 protein and Escherichia coli thioredoxin by a novel technique involving chromatography on Sephadex G-50 at pH 11.5. Analysis of the metal content of the holoenzyme by atomic absorption spectroscopy showed that it did not contain stoichiometric amounts of zinc. Determination of polymerase and exonuclease activities of the holoenzyme and the gene 5 protein in assay mixtures containing enzyme concentrations in excess of the Zn2+ concentration showed full activity. Addition of Zn2+ resulted in no stimulation and the activities were completely inhibited by 0.1 mM Zn2+. These results demonstrate that the essential T7 DNA polymerase is not a zinc-metalloenzyme and suggest that DNA polymerases show no functional requirement for Zn2+.
...
PMID:T7 DNA polymerase is not a zinc-metalloenzyme and the polymerase and exonuclease activities are inhibited by zinc ions. 643 10

The immunoadsorbent used to purify T7 DNA polymerase contains antibodies directed towards thioredoxin. Elution of the enzyme is made by a pulse of buffer at pH 12.0. This decreases the binding capacity of the column. Binding experiments with [3H]thioredoxin showed that the effect was caused by reduction of the antibodies by thiols in alkaline buffers. T7 DNA polymerase aggregated and irreversibly lost activity in buffers of low ionic strength. Experiments with gel chromatography and glycerol density gradient centrifugation showed that 0.2 M sodium chloride was required to keep the enzyme in its monomeric form. The sedimentation coefficient and the Stokes' radius are 5.3 S and 4.6 nm respectively, evaluated by gel chromatography and glycerol density gradient centrifugation techniques. The frictional ratio of 1.49 indicates that the T7 DNA polymerase is an asymmetrical protein.
...
PMID:An improved purification method and a physical characterization of phage T7 DNA polymerase. 675 19

T7 DNA polymerase reduced insulin at the same Km as thioredoxin, while the turnover number decreased. Recycling of the disulfide of thioredoxin subunit to its dithiol form was made by thioredoxin reductase. Incubation of T7 DNA polymerase with insulin decreases its ability to bind DNA and therefore inhibited polymerase and exonuclease activities. Thioredoxin reductase fully reversed this inhibition. Insulin did not induce dissociation of the T7 DNA polymerase subunits, which was tested by immunoadsorbent chromatography. No significant difference in single-stranded exonuclease compared to polymerase activity was seen in the flow through or the eluate, which had been expected if a dissociation of the subunits had occurred.
...
PMID:Inhibition of the T7 DNA polymerase by insulin. 676 Nov 43

DNA polymerase of bacteriophage T7 is composed of two subunits, the gene 5 protein of the phage and the host-coded thioredoxin. We have purified T7 DNA polymerase to homogeneity from T7-infected Escherichia coli B cells with a novel technique based on immunoadsorbent affinity chromatography. The enzyme binds quantitatively to a column of anti-thioredoxin Sepharose 4B and remains as an active complex in the immobilized state. It is eluted in fully active and highly purified form by a pulse of buffer at pH 12. After a final phosphocellulose chromatography, T7 DNA polymerase of better than 99% purity, as estimated from sodium dodecyl sulfate polyacrylamide gel electrophoresis, is obtained. Determination of the molecular weight by sedimentation equilibrium centrifugation gives a value of 112,000. Denaturing gels showed that the enzyme is composed of gene 5 protein (Mr = 87,000 +/- 3,000) and thioredoxin (Mr = 12,000) in a 1:1 stoichiometry. The amino acid composition of the enzyme and its spectrum was determined. The DNA polymerase activity is dependent on sulfhydryl compounds, sensitive to salt, and shows a comparatively high Km value for the four deoxyribonucleotides. The enzyme preparation has an inherent 3' leads to 5' exonuclease activity, attacking both native and denatured T7 DNA; it is free from detectable endonuclease activity.
...
PMID:Characterization of bacteriophage T7 DNA polymerase purified to homogeneity by antithioredoxin immunoadsorbent chromatography. 700 6

Thioredoxin was purified to homogeneity from the Escherichia coli mutant tsnC 7007 that is defective in phage T7 DNA replication and previously shown to contain a missense thioredoxin. Tryptic peptide maps of reduced and carboxymethylated 7007 thioredoxin combined with amino acid sequence analysis revealed one amino acid substitution; Gly-92 in thioredoxin is exchanged to an aspartic acid residue in the 7007 protein. The missense thioredoxin gave no activity with the gene 5 protein of phage T7 in the complementation to active T7 DNA polymerase. It competitively inhibited the complementation of wild type thioredoxin and gene 5 protein and formed a complex with the gene 5 protein that was retained by antithioredoxin Sepharose. The 7007 thioredoxin has reduced catalytic activity with thioredoxin reductase, ribonucleotide reductase, or as a protein disulfide reductase. The apparent Km value of 7007 thioredoxin as a substrate for thioredoxin reductase was increased 3-fold relative to normal thioredoxin, and the Vmax value was decreased 7-fold. The position of GLy-92 in the known three-dimensional structure of thioredoxin-S2 is correlated with the changed functional properties of the substituted mutant protein.
...
PMID:A mutant thioredoxin from Escherichia coli tsnC 7007 that is nonfunctional as subunit of phage T7 DNA polymerase. 700 7

The bacteriophage T7 induced DNA polymerase, consisting of the phage specified gene 5 protein associated with Escherichia coli thioredoxin, catalyzes the copolymerization of SP-dATP alpha S with dTTP, producing the alternating of polymer poly[dTs-A)] by a mechanism involving inversion of configuration at P alpha. Degradation of poly[d(5s-A)] by the nucleolytic action of E. coli DNA polymerase produced the dinucleotide pdTps-dA, whose configuration at the phosphorothioate diester was assigned as R by comparison of the phosphorus-31 nuclear magnetic resonance chemical shift (55.0 ppm downfield from H3PO4) with that of an authentic sample. Further degradation by alkaline phosphatase to Rp-dTps-dA (55.6 ppm downfield from H3PO4) confirmed the configuration. The stereochemistry provides no evidence of a double displacement mechanism.
...
PMID:Stereochemical course of nucleotidyl transfer catalyzed by bacteriophage T7 induced DNA polymerase. 709 4

Deletion of both thioredoxin genes TRX1 and TRX2 of Saccharomyces cerevisiae reduces the rate of DNA replication. This observation, originally determined by flow cytometry, was confirmed by radiochemical labeling of synchronized cultures. Since thioredoxin is a hydrogen donor to ribonucleotide reductase, a priori the inhibition of DNA synthesis was predicted to be caused by a reduction in the deoxyribonucleotide pools. However, the levels of TTP, dCTP, dATP, and dGTP were either unchanged or slightly greater in the thioredoxin mutant (3.2, 0.91, 1.4, and 1.21 pmol/10(6) cells, respectively) versus the wild-type culture (2.5, 0.91, 1.0, and 0.68 pmol/10(6) cells, respectively). An impact on ribonucleotide reduction was seen by an increased accumulation of RNR1 and RNR2 transcripts in the thioredoxin mutant (4.3- and 6.8-fold, respectively). Increased RNR expression did not reflect a general response of the DNA replication machinery. POL1 (DNA polymerase I) and CDC8 (thymidylate kinase) transcription were unaltered, while histone H2B transcripts actually decreased by half. Two alternative models incorporating these results are discussed. One suggests that thioredoxin reduces a multiprotein complex channeling nucleotides to the replication apparatus. The second proposes that thioredoxin regulates the tempo of DNA replication directly by activating a component of the replication machinery.
...
PMID:Deoxyribonucleotides are maintained at normal levels in a yeast thioredoxin mutant defective in DNA synthesis. 792 10

We have examined the effect of thioredoxin, an accessory protein that confers high processivity to bacteriophage T7 DNA polymerase, on the fidelity of DNA synthesis. In the presence of thioredoxin, exonuclease-proficient T7 DNA polymerase is highly accurate. In fidelity assays that score errors that revert M13mp2 lacZ alpha-complementation mutants, error rates are < or = 2.2 x 10(-6) for base substitution and < or = 3.7 x 10(-7) and < or = 4.5 x 10(-7) for frameshifts that revert mutations in the +1 and -1 reading frames, respectively. Rates are more than 10-fold higher during synthesis by polymerase.thioredoxin complex lacking 3'-->5' exonuclease activity, demonstrating that frameshift as well as substitution errors are subject to proofreading. The contribution of thioredoxin to accuracy has been examined by comparing the fidelity of the exonuclease-deficient polymerase in the presence or absence of the accessory protein. Thioredoxin either enhances or reduces fidelity, depending on the type of error considered. In the absence of thioredoxin, T7 DNA polymerase is 3-fold more accurate for base substitutions and > or = 27-fold and 9-fold more accurate, respectively, for 1- and 2-nt deletion errors at nonreiterated nucleotide sequences. Higher fidelity for all three errors may reflect the inability of the polymerase to continue synthesis from the premutational intermediates in the absence of the accessory protein. In marked contrast, the rate for frameshift errors wherein one or more nucleotides has been added to a repeated DNA sequence increases 46-fold when thioredoxin is absent from the polymerization reaction. The error rate increases as the length of the repeated sequence increases, consistent with a model where strand slippage creates misaligned template-primers. Thus, replicative expansion of repetitive sequences occurs in the absence of a replication accessory protein.
...
PMID:Error-prone replication of repeated DNA sequences by T7 DNA polymerase in the absence of its processivity subunit. 804 4


<< Previous 1 2 3 4 5 6 7 8 Next >>

---