Characterization of bacteriophage T7 DNA polymerase purified to homogeneity by antithioredoxin immunoadsorbent chromatography.

Nordström, B; Randahl, Håkan; Slaby, Ivan; Holmgren, Arne

doi:10.1016/s0021-9258(19)69731-0

Cited by 35 publications

(4 citation statements)

References 19 publications

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“…The results shown in Table I compare the DNA polymerases from Thermococcus litoralis and Thermus flavis to T7 DNA polymerase, an enzyme having an associated 3'-5' exonuclease activity (18,19,20). Primer extension by T7 DNA polymerase creates a product yielding 6.0-8.6% medium-blue plaques, suggesting that the mismatch in the primer terminus is excised as expected prior to synthesis by T7 DNA polymerase under stated reaction conditions.…”

Section: Resultsmentioning

confidence: 98%

Fidelity of DNA synthesis by the Thermococcus litoralis DNA polymerase—an extremely heat stable enzyme with proofreading activity

Mattila¹,

Korpela²,

Tenkanen³

et al. 1991

Nucleic Acids Research

217

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We demonstrate that the DNA polymerase isolated from Thermococcus litoralis (VentTM DNA polymerase) is the first thermostable DNA polymerase reported having a 3'----5' proofreading exonuclease activity. This facilitates a highly accurate DNA synthesis in vitro by the polymerase. Mutational frequencies observed in the base substitution fidelity assays were in the range of 30 x 10(-6). These values were 5-10 times lower compared to other thermostable DNA polymerases lacking the proofreading activity. All classes of DNA polymerase errors (transitions, transversions, frameshift mutations) were assayed using the forward mutational assay (1). The mutation frequencies of Thermococcus litoralis DNA polymerase varied between 15-35 x 10(-4) being 2-4 times lower than the respective values obtained using enzymes without proofreading activity. We also noticed that the fidelity of the DNA polymerase from Thermococcus litoralis responds to changes in dNTP concentration, units of enzyme used per one reaction and the concentration of MgSO4 relative to the total concentration of dNTPs present in the reaction. The high fidelity DNA synthesis in vitro by Thermococcus litoralis DNA polymerase provides good possibilities for maintaining the genetic information of original target DNA sequences intact in the DNA amplification applications.

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Section: Resultsmentioning

confidence: 98%

Fidelity of DNA synthesis by the Thermococcus litoralis DNA polymerase—an extremely heat stable enzyme with proofreading activity

Mattila¹,

Korpela²,

Tenkanen³

et al. 1991

Nucleic Acids Research

217

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“…The mutant thioredoxins behaved similarly to the wild-type protein during purification. The E. coli Trx from the host strain was removed efficiently by E. coli Trx antibody affinity chromatography (Nordstrom et al, 1981). In the wild-type or mutant protein solutions the activity of the E. coli Trx contaminant was not detectable using a sensitive insulin The assay mixture contained 100 mM potassium phosphate, pH 7,0, 2 mM EDTA, 0.16 mM insulin, and 0.15 mM NADPH.…”

Section: Resultsmentioning

confidence: 99%

Mutagenesis of structural half-cystine residues in human thioredoxin and effects on the regulation of activity by selenodiglutathione

Ren¹,

Björnstedt²,

Shen³

et al. 1993

Biochemistry

151

105

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A human thioredoxin cDNA was modified to optimize Escherichia coli expression and subcloned into the plasmid pACA, a vector for T7 RNA polymerase-directed expression. The substitution of structural (noncatalytic) half-cystines in human thioredoxin (hTrx) was made by site-directed mutagenesis. The recombinant wild-type (wt) hTrx and its mutant C61S, C72S, and C61S/C72S were expressed and purified to homogeneity. Characterization of the wt and mutant hTrx was done with respect to redox activity with thioredoxin reductase (TR), tryptophan fluorescence, and effects of incubation with GS-Se-SG, which is believed to be the major metabolite of inorganic selenium compounds in mammalian tissues. The Km and kcat of wild-type hTrx for human placenta thioredoxin reductase (HP-TR) at pH 7.0 were 2.0 microM and 2800 min-1, respectively. The mutant proteins C61S, C72S, and C61S/C72S had Km and kcat values similar to those of the wt thioredoxin. Tryptophan fluorescence measurements showed that the wt and mutant proteins had similar stability to a denaturing agent. Incubation of fully reduced thioredoxin with 0.1 molar equivalent of GS-Se-SG resulted in continued oxidation of SH groups. After 3.5 h only 0.5 of initially 4.6 SH groups/thioredoxin remained. With the oxidized protein, a pronounced lag phase in thioredoxin reductase-dependent insulin disulfide reduction was present. Disulfide-linked dimers of the protein were present. The results clearly showed that noncatalytic cysteine residues in hTrx were oxidized accompanied by dimerization and inactivation. The activities of the mutant proteins C72S and C61S/C72S were unchanged after 3 h of incubation with GS-Se-SG. No dimer appeared of the C72S thioredoxin.(ABSTRACT TRUNCATED AT 250 WORDS)

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“…This is significant and shows that the structure of the molecule outside the local hydrophobic pocket that contains these two charged side chains is unaffected by the mutations. The specificity of the binding for the reduced form of thioredoxin initially suggested that the active site thiols were involved in the binding, but later work Huber et al, 1986) showed that the cysteine thiols themselves were not absolutely required, and that the loops containing Gly 92 and Ile 75 were most important for this reaction (Nordstro ¨m et al, 1981). We have recently suggested (Stone et al, 1993; that the difference between the two forms of thioredoxin in binding to gene 5 protein is due to the greater relative mobility of the polypeptide chain in this region of Trx-(SH) 2 , which allows the molecule to bind to the G5p in a conformation other than the ground state structure found in solution, which is practically identical to that of Trx-S 2 (Jeng et al, 1994).…”

Section: Effects Of the Mutations On The Structure Of Thioredoxinmentioning

confidence: 99%

Effects of Buried Charged Groups on Cysteine Thiol Ionization and Reactivity in Escherichia coli Thioredoxin: Structural and Functional Characterization of Mutants of Asp 26 and Lys 57

et al. 1997

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To investigate the role of Asp 26 and Lys 57, two conserved, buried residues, in the redox mechanism of Escherichia coli thioredoxin (Trx), three mutant proteins, Asp 26 --> Ala (D26A), Lys 57 --> Met (K57M), and the double mutant D26A/K57M, were prepared, replacing the charged amino acids with hydrophobic residues with similar sizes. Both the oxidized (Trx-S2) and reduced [Trx-(SH)2] forms of the mutant thioredoxins are fully folded and similar in overall structure to the wild-type protein (wt). The structure of the active site hydrophobic surface is unchanged by the mutation of Asp 26 and Lys 57, since DNA polymerase activity in the 1:1 complex of the T7 gene 5 protein and mutant Trx-(SH)2 shows similar Kd values (approximately 5 nM) for both mutants and wt. In contrast, redox reactions involving thioredoxin as a catalyst of the reduction of disulfides or oxidation of dithiols are strongly affected by the mutations. In the reaction of Trx-S2 with thioredoxin reductase at pH 8.0, the kcat/Km value for the D26A mutant is decreased by a factor of 10 from that of wt, while the value for the D26A/K57M mutant is reduced 40-fold. The activity of Trx-(SH)2 as a protein disulfide reductase was measured with insulin, using fluorescence to detect oxidation of thioredoxin. At 15 degrees C and pH 8.0, both the D26A and K57M mutants showed 5--10-fold decreases in rates of reaction compared to those of the wild type, and the pH-rate profiles for the mutants were shifted 1 (K57M) and 2 (D26A) units to higher pH compared with the wt curve. NMR measurements for the three mutant proteins indicate that the proteins have the same global fold as that of the wild type, although changes in the chemical shifts of a number of resonances indicate local structural changes in the active site region. The resonances of oxidized D26A and D26A/K57M are pH-independent between pH 6.0 and 10.0, confirming the identification of the active site group titrating with a pKa of 7.5 in wt Trx-S2 as Asp 26. A profound change in the pKa of Asp 26, from 7.5 in the wild type to 9.4 in the mutant, is observed for K57M Trx-S2. The pH-dependent behavior of the resonances is affected in all mutant Trx-(SH)2 proteins. A single pKa shifted to higher values is observed on both the Cys 32 and Cys 35 Cbeta resonances. Ultraviolet absorbance measurements (A240) as a function of pH for wt Trx-(SH)2 demonstrate that the cysteine thiols titrate with apparent pK(a)s of about 7.1 and 9.9. The mutant proteins each show a single transition in the A240 measurements, with a midpoint at pH 7.8-8.0, consistent with the NMR results. The change in absorbance at 240 nm with increasing pH indicates that the number of thiols titrating in each mutant is greater than one but less than two. It is clear that both thiol pK(a)s have been significantly shifted by the mutations. The Cys 32 pKa is moved from 7.1 in wt to 7.8-8.0 in the mutants. The value of the Cys 35 pKa either is indistinguishable from that of Cys 32, thus accounting for more than one thiol titrating in the UV absorbance m...

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Characterization of bacteriophage T7 DNA polymerase purified to homogeneity by antithioredoxin immunoadsorbent chromatography.

Cited by 35 publications

References 19 publications

Fidelity of DNA synthesis by the Thermococcus litoralis DNA polymerase—an extremely heat stable enzyme with proofreading activity

Fidelity of DNA synthesis by the Thermococcus litoralis DNA polymerase—an extremely heat stable enzyme with proofreading activity

Mutagenesis of structural half-cystine residues in human thioredoxin and effects on the regulation of activity by selenodiglutathione

Effects of Buried Charged Groups on Cysteine Thiol Ionization and Reactivity in Escherichia coli Thioredoxin: Structural and Functional Characterization of Mutants of Asp 26 and Lys 57

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