EFFECT OF REACTION PH ON THE FIDELITY AND PROCESSIVITY OF EXONUCLEASE-DEFICIENT KLENOW POLYMERASE

We have examined as a function of pH the fidelity of DNA synthesis cat alyzed by the 3'-->5' exonuclease-deficient form of the Klenow fragmen t of Escherichia coli DNA polymerase I. Increasing the pH of in vitro gap-filling reactions from pH 6.2 through 9.8 (37-degrees-C increased the frequency of base substitution and minus-one-base frameshift mutat ions 50- and 40-fold, respectively, as measured by reversion of a nons ense or frameshift mutation within the lacZalpha gene of bacteriophage M13mp2. To understand the mechanisms of high fidelity at low pH, we h ave examined the biochemical events associated with DNA synthesis at p H 6.2 that might be responsible for the observed accuracy in vitro. We show that while the steady-state frequency of T.dGTP misinsertion at the lacZalpha opal codon is 20-fold lower at pH 6.2 than at pH 7.6, pH -dependent changes in the frequencies of G.dATP and A.dCTP base misins ertions at the lacZalpha nonsense codon are insufficient to explain th e fidelity changes observed in the gap-filling assay. However, the eff iciency of steady-state extension synthesis from template-primers cont aining 3'-terminal T.G, G.A, and A.C (template-primer) mispairs was re duced up to 160-fold at pH 6.2 relative to pH 7.6. Analyses of the pro cessivity of DNA polymerization versus pH demonstrated that at low pH the termination probability was decreased at specific template positio ns. Concomitantly, at sites where the termination probability was lowe r at pH 6.2, a decreased error rate was observed for base substitution mutations at three template positions and for minus-one-base frameshi ft mutations at two homopolymeric sequences relative to pH 7.6. We sug gest that the observed increase in error discrimination by the exonucl ease-deficient Klenow polymerase results from altered template binding properties of the enzyme at pH 6.2.