Effect of reaction pH on the fidelity and processivity of exonuclease-deficient Klenow polymerase.

Abstract:

We have examined as a function of pH the fidelity of DNA synthesis catalyzed by the 3'-->5' exonuclease-deficient form of the Klenow fragment 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 mutations 50- and 40-fold, respectively, as measured by reversion of a nonsense or frameshift mutation within the lacZ alpha gene of bacteriophage M13mp2. To understand the mechanisms of high fidelity at low pH, we have examined the biochemical events associated with DNA synthesis at pH 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 lacZ alpha 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 misinsertions at the lacZ alpha nonsense codon are insufficient to explain the fidelity changes observed in the gap-filling assay. However, the efficiency of steady-state extension synthesis from template-primers containing 3'-terminal T.G, G.A, and A.C (template-primer) mispairs was reduced up to 160-fold at pH 6.2 relative to pH 7.6. Analyses of the processivity of DNA polymerization versus pH demonstrated that at low pH the termination probability was decreased at specific template positions. Concomitantly, at sites where the termination probability was lower at pH 6.2, a decreased error rate was observed for base substitution mutations at three template positions and for minus-one-base frameshift mutations at two homopolymeric sequences relative to pH 7.6. We suggest that the observed increase in error discrimination by the exonuclease-deficient Klenow polymerase results from altered template binding properties of the enzyme at pH 6.2.

Polymerases:

Topics:

Exonuclease Activity, Fidelity, Source / Purification, Nucleotide Incorporation

Status:

new topics/pols set partial results complete validated

Results:

Polymerase Reference Property Result Context
Klenow (exo-) Eckert KA1993 3-5' Exonuclease (proofreading) No
Klenow (exo-) Eckert KA1993 Frameshift Error Rate 1.4E-05 errors/bp
Klenow (exo-) Eckert KA1993 Frameshift Error Rate 0.00054 errors/bp
Klenow (exo-) Eckert KA1993 Nucleotide Substitution Rate 1.4E-05 errors/bp Technique: Reversion (pH 6.2)
Klenow (exo-) Eckert KA1993 Nucleotide Substitution Rate 0.00076 errors/bp Technique: Reversion (pH 9.8)
Klenow (exo-) Eckert KA1993 Full length or truncated Full length
Klenow (exo-) Eckert KA1993 Gap Filling Yes

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