Amino acid changes in a unique sequence of bacteriophage T7 DNA polymerase alter the processivity of nucleotide polymerization.

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.

Polymerases:

Topics:

Nucleotide Incorporation, Source / Purification

Status:

new topics/pols set partial results complete validated

Results:

Polymerase Reference Property Result Context
T7 Yang XM1997 Full length or truncated Full length
T7 Yang XM1997 Processivity 7000bp
T7 (no thioreoxin) Yang XM1997 Full length or truncated Full length
T7 (no thioreoxin) Yang XM1997 Processivity 50bp

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