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)

A mutant of Escherichia coli thioredoxin containing serine residues in place of the two active-site cysteines, termed C32S,C35S, previously shown to be partially able to substitute for reduced thioredoxin in certain phage systems, has been characterized by 1H NMR spectroscopy at pH values between 5.5 and 10. The 1H NMR spectrum of the mutant at pH 5.5 is very similar to that of the wild-type protein in either the reduced or oxidized state. Chemical shift changes in the vicinity of the active site serines indicate that the nearby hydrophobic pocket is somewhat changed, probably as a result of the replacement of the cysteine thiols with the smaller, more hydrophilic hydroxyl side chains and a change in the preferred chi 1 angles of the side chains. Although the pattern of amide protons persistent in 2H2O differs only slightly between the two forms of the wild-type protein, the pattern observed for the C32S,C35S mutant shows characteristic features that correspond closely with those of the reduced wild-type protein rather than with the oxidized form. The pH dependence of the mutant protein shows a single group titrating close to the active site with a pKa of 8.3, which we assign to the buried carboxyl group of Asp 26 by analogy with the behavior of wild-type thioredoxin. The pKa is significantly higher for the mutant protein, consistent with an increase in the hydrophobicity of the pocket where the carboxyl is buried, probably due to repacking caused by the removal of the cysteine thiols and the placement of the serine hydroxyls in positions where they interact better with solvent. The results demonstrate that the solution behavior of the mutant protein is similar in many ways to that of reduced wild-type thioredoxin, explaining its partial activity in the two essential roles of reduced thioredoxin as a subunit of phage T7 DNA polymerase and in the assembly of filamentous phage.
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PMID:Characterization by 1H NMR of a C32S,C35S double mutant of Escherichia coli thioredoxin confirms its resemblance to the reduced wild-type protein. 831 57

We have produced several mutants of Escherichia coli thioredoxin (Trx) using a combined mutagenesis/chemical modification technique. The protein C32S, C35S, L78C Trx was produced using standard mutagenesis procedures. After unfolding the protein with guanidine hydrochloride (GdmCl), the normally buried cysteine residue was modified with a series of straight chain aliphatic thiosulfonates, which produced cysteine disulfides to methane, ethane, 1-n-propane, 1-n-butane, and 1-n-pentane thiols. These mutants all show native-like CD spectra and the ability to activate T7 gene 5 protein DNA polymerase activity. In addition, all mutants show normal unfolding transitions in GdmCl solutions. However, the midpoint of the transition, [GdmCl]1/2, and the free energy of unfolding at zero denaturant concentration, delta G(H2O), give inverse orders of stability. This effect is due to changes in m, the dependence of delta G0 unfolding on the GdmCl concentration. The method described here may be used to produce unnatural amino acids in the hydrophobic cores of proteins.
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PMID:Unnatural amino acid packing mutants of Escherichia coli thioredoxin produced by combined mutagenesis/chemical modification techniques. 845 77

Escherichia coli thioredoxin contains two tryptophan residues (Trp28 and Trp31) situated close to the active site disulfide/dithiol. In order to probe the structural and functional roles of tryptophan in the mechanism of E. coli thioredoxin (Trx), we have replaced Trp28 with alanine using site-directed mutagenesis and expressed the mutant protein W28A in E. coli. Changes in the behavior of the mutant protein compared with the wild-type protein have been monitored by a number of physical and spectroscopic techniques and enzyme assays. As expected, removal of a tryptophan residue causes profound changes in the fluorescence spectrum of thioredoxin, particularly for the reduced protein (Trx-(SH)2), and to a lesser extent for the oxidized protein (Trx-S2). These results show that the major contribution to the strongly quenched fluorescence of Trx-S2 in both wild-type and mutant proteins is from Trp31, whereas the higher fluorescence quantum yield of Trx-(SH)2 in the wild-type protein is dominated by the emission from Trp28. The fluorescence, CD, and 1H NMR spectra are all indicative that the mutant protein is fully folded at pH 7 and room temperature, and, despite the significance of the change, from a tryptophan in close proximity to the active site to an alanine, the functions of the protein appear to be largely intact. W28A Trx-S2 is a good substrate for thioredoxin reductase, and W28A Trx-(SH)2 is as efficient as wild-type protein in reduction of insulin disulfides. DNA polymerase activity exhibited by the complex of phage T7 gene 5 protein and Trx-(SH)2 is affected only marginally by the W28A substitution, consistent with the buried position of Trp28 in the protein. However, the thermodynamic stability of the molecule appears to have been greatly reduced by the mutation: guanidine hydrochloride unfolds the protein at a significantly lower concentration for the mutant than for wild type, and the thermal stability is reduced by about 10 degrees C in each case. The stability of each form of the protein appears to be reduced by the same amount, an indication that the effect of the mutation is identical in both forms of the protein. Thus, despite its close proximity to the active site, the Trp28 residue of thioredoxin is not apparently essential to the electron transfer mechanism, but rather contributes to the stability of the protein fold in the active site region.
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PMID:Replacement of Trp28 in Escherichia coli thioredoxin by site-directed mutagenesis affects thermodynamic stability but not function. 862 6

We identified and sequenced a gene encoding a third thioredoxin (C3) from Corynebacterium nephridii. The determined nucleotide sequence encodes a thioredoxin of 145 amino acid residues, which is larger than most thioredoxins found in microbial cells and contains 6 cysteine residues. C. nephridii thioredoxin C3 is able to serve as a subunit of T7 DNA polymerase. C. nephridii is the first nonphotosynthetic procaryotic organism known to carry three different thioredoxins.
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PMID:Identification of a third thioredoxin gene from Corynebacterium nephridii. 865 61

In this study, we report the effects of two different substitutions in Rhodobacter sphaeroides thioredoxin on two regions of the protein: the N-terminus end and the hydrophobic area implicated in protein/protein interactions. We have produced by site-directed mutagenesis R. sphaeroides thioredoxin single and double mutants in which the glycine residue at position 74 is changed to a serine and the serine at position 3 is changed to an alanine; the three mutant proteins have been purified. The two substitutions are not equivalent. Substitution of serine by alanine increased the pI from 5.2 to 6.1; this pI value was the same in the double-mutated protein, which demonstrates the presence of a local conformational change. In vivo studies showed that the Gly74-->Ser substitution completely prevented phage T3/T7 growth whereas the Ser3-->Ala substitution had no effect. This finding was corroborated by the large decrease (100-fold) of polymerase activity for the double mutant in the in vitro measurement of phage T7 DNA polymerase activity with the corresponding pure proteins. Although marginal (within a factor of two), the effects of the two substitutions on the catalytic activities of the thioredoxin reductase reaction confirmed their difference. Substitution of serine by alanine had no effect on the Km and resulted in an improvement in the catalytic efficiency. In contrast, the second substitution increased the Km value, without improving the catalytic efficiency. The following can be concluded (a) glycine74 of R. sphaeroides thioredoxin has a direct role in the binding of T7 gene 5 protein and the hydrophobic area of thioredoxin; (b) the N-terminus plays a role in maintaining the conformational integrity of the active site; (c) the flexibility of Gly74 in the hydrophobic region involved in protein/protein interaction is the operative factor in the case of the activity of thioredoxin in the T7 DNA polymerase.
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PMID:The Gly74-->Ser and Ser3-->Ala mutations in Rhodobacter sphaeroides Y thioredoxin: effects on active site reactivity and protein interaction. 865 21

Upon infection of Escherichia coli, bacteriophage T7 annexes a host protein, thioredoxin, to serve as a processivity factor for its DNA polymerase, T7 gene 5 protein. In a previous communication (Himawan, J., and Richardson, C. C. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 9774-9778), we reported that an E. coli strain encoding a Gly-74 to Asp-74 (G74D) thioredoxin mutation could not support wild-type T7 growth and that in vivo, six mutations in T7 gene 5 could individually suppress this G74D thioredoxin defect. In the present study, we report the purification and biochemical characterization of the G74D thioredoxin mutant and two suppressor gene 5 proteins, a Glu-319 to Lys-319 (E319K) mutant of gene 5 protein and an Ala-45 to Thr-45 (A45T) mutant. The suppressor E319K mutation, positioned within the DNA polymerization domain of gene 5 protein, appears to suppress the parental thioredoxin mutation by compensating for the binding defect that was caused by the G74D alteration. We suggest that the Glu-319 residue of T7 gene 5 protein and the Gly-74 residue of E. coli thioredoxin define a contact point or site of interaction between the two proteins. In contrast, the A45T mutation in gene 5 protein, located within the 3' to 5' exonuclease domain, does not suppress the G74D thioredoxin mutation by simple restoration of binding affinity. Based upon our understanding of the mechanisms of suppression, we propose a model for the T7 gene 5 protein-E. coli thioredoxin interaction.
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PMID:Amino acid residues critical for the interaction between bacteriophage T7 DNA polymerase and Escherichia coli thioredoxin. 870 17

The 80-kDa gene 5 protein encoded by bacteriophage T7 shares significant amino acid homology with the large fragment of Escherichia coli DNA polymerase I (Klenow fragment). Like the Klenow fragment, T7 gene 5 protein has both DNA polymerase and 3' to 5' exonuclease activities. However, unlike the Klenow fragment, T7 gene 5 protein binds tightly to E. coli thioredoxin to form a complex that has a high processivity of nucleotide polymerization. In order to identify the domains of gene 5 protein responsible for polymerization, hydrolysis, and binding of thioredoxin, we have analyzed proteolytic fragments of gene 5 protein. Cleavage of the protein within one protease-sensitive region (residue 250-300) yields two molecular weight species of peptides of 32-37 and 43-51 kDa derived from the N-terminal and C-terminal region, respectively. DNA polymerase activity is found within the C-terminal fragments and exonuclease activity within the N-terminal fragments. Thioredoxin stimulates the DNA polymerase activity of the C-terminal fragments. All fragments bind to DNA. In addition to delineating the polymerase and exonuclease domains, the protease-sensitive region appears to interact with E. coli thioredoxin. Thioredoxin protects this region from proteolysis, and alteration of this region reduces the ability of thioredoxin to stimulate polymerase activity.
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PMID:Structural and functional organization of the DNA polymerase of bacteriophage T7. 879 63

In Klenow fragment DNA polymerase, a flexible 50-amino acid subdomain at the tip of the thumb which includes two alpha helices has been suggested to interact with the duplex template-primer (Beese, L.S., Derbyshire, V. and Steitz, T.A. (1993) Science 260, 352-355). The present study investigates the properties of Klenow polymerase containing a 24-amino acid deletion (residues 590-613) that removes a portion of the tip of the thumb. The mutant polymerase has relatively normal dNTP binding and catalytic rate. However, its DNA binding affinity is reduced by more than 100-fold relative to the intact polymerase and its ability to conduct processive synthesis is also reduced. Although the mutant polymerase has relatively normal base substitution fidelity, it has strongly reduced frameshift fidelity, being especially error-prone for single nucleotide addition errors in homopolymeric runs. The addition error rate increases as the length of the reiterated sequence increases, indicative of errors initiated by template-primer strand slippage. These observations suggest a role for the tip of the thumb of Klenow polymerase in determining DNA binding, processivity and frameshift fidelity, perhaps by tracking the minor groove of the duplex DNA. The results are discussed in light of remarkably similar observations with T7 DNA polymerase in the presence or absence of thioredoxin, an accessory subunit that affects these same properties.
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PMID:A thumb subdomain mutant of the large fragment of Escherichia coli DNA polymerase I with reduced DNA binding affinity, processivity, and frameshift fidelity. 879 75

Bacteriophage T7 DNA polymerase shares extensive sequence homology with Escherichia coli DNA polymerase I. However, in vivo, E. coli DNA polymerase I is involved primarily in the repair of DNA whereas T7 DNA polymerase is responsible for the replication of the viral genome. In accord with these roles, T7 DNA polymerase is highly processive while E. coli DNA polymerase I has low processivity. The high processivity of T7 DNA polymerase is achieved through tight binding to its processivity factor, E. coli thioredoxin. We have identified a unique 76-residue domain in T7 DNA polymerase responsible for this interaction. Insertion of this domain into the homologous site in E. coli DNA polymerase I results in a dramatic increase in the processivity of the chimeric DNA polymerase, a phenomenon that is dependent upon its binding to thioredoxin.
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PMID:The thioredoxin binding domain of bacteriophage T7 DNA polymerase confers processivity on Escherichia coli DNA polymerase I. 901 9

T7 gene 5 DNA polymerase forms a complex with Escherichia coli thioredoxin (its processivity factor), and a 76-amino acid sequence (residues 258-334), unique to gene 5 protein, has been implicated in this interaction. We have examined the effect of amino acid substitution(s) in this region on T7 phage growth and on the interaction of the polymerase with thioredoxin. Among the mutations in gene 5, we found that a substitution of either Glu or Ala for Lys-302 yielded a protein that could not complement T7 phage lacking gene 5 (T7Delta5) to grow on E. coli having reduced thioredoxin levels. One triple mutant (K300E,K302E,K304E) could not support the growth of T7Delta5 even in wild type cells. This altered polymerase is stimulated 4-fold less by thioredoxin than is the wild type enzyme and the polymerase-thioredoxin complex has reduced processivity. The exonuclease activity of the altered polymerase is not stimulated to the same extent as that of the wild type enzyme by thioredoxin. The observed dissociation constant of the gene 5 protein K(300,302,304)E-thioredoxin complex is 7-fold higher than that of the wild type complex. The altered polymerase also has a lower binding affinity for double-stranded DNA.
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PMID:Amino acid changes in a unique sequence of bacteriophage T7 DNA polymerase alter the processivity of nucleotide polymerization. 904 89


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