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 thiored oxin (its processivity factor), and a 76-amino acid sequence (residues 258-334), unique to gene 5 protein, has been implicated in this inter action. We have examined the effect of amino acid substitution(s) in t his region on T7 phage growth and on the interaction of the polymerase with thioredoxin. Among the mutations in gene 5, we found that a subs titution of either Glu or Ala for Lys-302 yielded a protein that could not complement T7 phage lacking gene 5 (T7 Delta 5) to grow on E. col i having reduced thioredoxin levels. One triple mutant (K300E,K302E,K3 04E) could not support the growth of T7 Delta 5 even in wild type cell s. This altered polymerase is stimulated 4-fold less by thioredoxin th an is the wild type enzyme and the polymerase thioredoxin complex has reduced processivity. The exonuclease activity of the altered polymera se is not stimulated to the same extent as that of the wild type enzym e by thioredoxin. The observed dissociation constant of the gene 5 pro tein 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 bindin g affinity for double-stranded DNA.