Mechanisms of error discrimination by Escherichia coli DNA polymerase I

Abstract

The mechanism of base selection by DNA polymerase I of Escherichia coli has been investigated by kinetic analysis. The apparent KM for the insertion of the complementary nucleotide dATP into the hook polymer poly(dT)-oligo(dA) was found to be 6-fold lower than that for the noncomplementary nucleotide dGTP, whereas the Vmax for insertion of dATP was 1600-fold higher that that for dGTP. The ratio of Kcat/KM values for complementary and mismatched nucleotides of 104 demonstrates the extremely high specificity of base selection by DNA polymerase I is in agreement with results obtained with a different template-primer, poly(dC)-oligo(dG) [El-Deiry, W. S., Downey, K. M., and So, A. G. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 7378]. Studies on the effects of phosphate ion on the polymerase and 3''- to 5''-exonuclease activities of DNA polymerase I showed that, whereas the polymerase activity was somewhat stimulated by phosphate, the exonuclease activity was markedly inhibited, being 50% inhibited at 25 nM phosphate and greater than 90% inhibited at 80 mM phosphate. Selective inhibition of the exonuclease activity by phosphate also resulted in inhibition of template-dependent conversion of a noncomplementary dNTP to dNMP and, consequently, markedly affected the kinetic constants for insertion of noncomplementary nucleotides. The mutagenic metal ion Mn2+ was found to affect error discrimination by both the polymerase and 3''- and 5''-exonuclease activities of DNA polymerase I. Substitution of Mn2+ for Mg2+ resulted in both decreased specificity of base selection by the polymerase activity and decreased specificity of proofreading by the 3''- to 5''-exonuclease activity. Furthermore, in the presence of Mn2+ but not Mg2+, mismatched primer termini could be extended by the polymerase, resulting in an increased error frequency. In addition to lowering the proofreading specific of the 3''- to 5''-exonuclease, Mn2+ also increased the sensitivity of the 3''- to 5''-exonuclease to selective inhibition by nucleotide 5''-monophosphates (5''NMPs). Thus, Mn2+-induced mutagenesis may be due not only to the relaxed specificity of the polymerase in base selection and the relaxed specificity of the 3''- to 5''-exonuclease in proofreading but also to increased sensitivity of proofreading to inhibition by 5''NMP. We have demonstrated that template-dependent turnover of noncomplementary dNTP to dNMP requires a 3''-hydroxyl group at the primer terminus and can be abolished by inhibition of the 3''- to 5''-exonuclease activity of the enzyme. These data suggest that "kinetic proofreading" does not play a major role in maintaining the accuracy of DNA synthesis with DNA polymerase I.