Amino acid changes in a unique sequence of bacteriophage T7 DNA polymerase alter the processivity of nucleotide polymerization.
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.
Yang
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Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA.
Richardson
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D017123:Bacteriophage T7 / Q000201:enzymology*; D001665:Binding Sites; D004247:DNA / Q000096:biosynthesis*; D004252:DNA Mutational Analysis; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry / Q000378:metabolism*; D005090:Exodeoxyribonucleases / Q000378:metabolism; D005816:Genetic Complementation Test; D013329:Structure-Activity Relationship; D013879:Thioredoxins / Q000378:metabolism
D013879:Thioredoxins; D004247:DNA; C034970:bacteriophage T7 induced DNA polymerase; D004259:DNA-Directed DNA Polymerase; D005090:Exodeoxyribonucleases