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Amino acid changes in a unique sequence of bacteriophage T7 DNA polymerase alter the processivity of nucleotide
1Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA.
Abstract:
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.
Insights
Mutations in T7 gene 5 DNA polymerase affect its interaction with Escherichia coli thioredoxin. This impacts T7 phage growth, particularly in cells with low thioredoxin levels.
Area of Science:
- Molecular Biology
- Virology
- Biochemistry
Background:
- T7 gene 5 DNA polymerase interacts with Escherichia coli thioredoxin, a processivity factor.
- A specific 76-amino acid region (residues 258-334) in gene 5 protein is crucial for this interaction.
Purpose of the Study:
- To investigate the impact of amino acid substitutions in the T7 gene 5 protein's thioredoxin-binding region.
- To assess how these mutations affect T7 phage growth and the polymerase-thioredoxin interaction.
Main Methods:
- Site-directed mutagenesis of T7 gene 5.
- Complementation assays for T7 phage growth in E. coli with varying thioredoxin levels.
- Biochemical analysis of polymerase-thioredoxin complex formation, processivity, and DNA binding affinity.
Main Results:
- Specific mutations (e.g., Glu or Ala for Lys-302) impaired T7 phage growth in low-thioredoxin E. coli.
- A triple mutant (K300E,K302E,K304E) failed to support T7 phage growth even in wild-type cells.
- The altered polymerase showed reduced stimulation by thioredoxin, decreased complex processivity, altered exonuclease activity stimulation, increased thioredoxin dissociation constant, and lower double-stranded DNA binding affinity.
Conclusions:
- The 76-amino acid region of T7 gene 5 protein is critical for efficient thioredoxin interaction and T7 phage propagation.
- Disrupting this interaction significantly impacts polymerase function and viral growth, highlighting the importance of the polymerase-thioredoxin complex.