Studeis on the biochemical basis of mutation. IV. Effect of amino acid substitution on the enzymatic and biological properties of bacteriophage T4 DNA polymerase

The effects of substituting specific amino acids at specified loci in the bacterio-phage T4 DNA polymerase molecule have been studied. Gene 43 (DNA polymerase) amber mutants grown on suppressor strains which substitute serine, glutamine, or tyrosine at specific sites in the polymerase molecule, produce enzymes with substantially different physical, enzymatic and biological properties when compared to wild type. When amB22, a gene 43 mutant which makes a DNA polymerase fragment with only 3′-exonuclease activity, was grown in Escherichia coli B40(sup⁺1), -(sup⁺ 2) or -(sup⁺3), enzymes with different temperature sensitivities and nuclease to polymerase ratios were produced. Measurements of spontaneous mutation rates in these suppressed strains indicated that the two with higher than normal exonuclease activity were antimutators, and the one with a slightly lower exonuclease activity was a mutator. The substituted amino acids at the amB22 site perturbed the 3′-exonuclease activity creating either antimutator or mutator phenotypes. Thus, the B22 enzymes provide additional biochemical evidence to support the hypothesis that the exonuclease to polymerase ratio may influence the spontaneous mutation rate in phage T4.