DNA polymerase insertion fidelity. Gel assay for site-specific kinetics.

Abstract:

A quantitative assay based on gel electrophoresis is described to measure nucleotide insertion kinetics at an arbitrary DNA template site. The assay is used to investigate kinetic mechanisms governing the fidelity of DNA synthesis using highly purified Drosophila DNA polymerase alpha holoenzyme complex and M13 primer-template DNA. Km and Vmax values are reported for correct insertion of A and misinsertion of G, C, and T opposite a single template T site. The misinsertion frequencies are 2 X 10(-4) for G-T and 5 X 10(-5) for both C-T and T-T relative to normal A-T base pairs. The dissociation constant of the polymerase-DNA-dNTP complex, as measured by Km, plays a dominant role in determining the rates of forming right and wrong base pairs. Compared with Km for insertion of A opposite T (3.7 +/- 0.7 microM), the Km value is 1100-fold greater for misinsertion of G opposite T (4.2 +/- 0.4 mM), and 2600-fold greater for misinsertion of either C or T opposite T (9.8 +/- 4.2 mM). These Km differences indicate that in the enzyme binding site the stability of A-T base pairs is 4.3 kcal/mol greater than G-T pairs and 4.9 kcal/mol greater than C-T or T-T pairs. In contrast to the large differences in Km, differences in Vmax are relatively small. There is only a 4-fold reduction in Vmax for insertion of G opposite T and an 8-fold reduction for C or T opposite T, compared with the correct insertion of A. For the specific template T site investigated, the nucleotide insertion fidelity for Drosophila polymerase alpha seems to be governed primarily by a Km discrimination mechanism.

Polymerases:

Topics:

Kinetic Parameters

Status:

new topics/pols set partial results complete validated

Results:

Polymerase Reference Property Result Context
Human Pol alpha Boosalis MS1987 KM 3.7uM Reaction: Nucleotide incorporation; Substrate: dATP; Technique: Gel shift
Human Pol alpha Boosalis MS1987 KM 4200uM Reaction: Nucleotide incorporation; Substrate: dGTP; Technique: Gel shift
Human Pol alpha Boosalis MS1987 KM 1E+04uM Reaction: Nucleotide incorporation; Substrate: dTTP; Technique: Gel shift
Human Pol alpha Boosalis MS1987 KM 1E+04uM Reaction: Nucleotide incorporation; Substrate: dCTP; Technique: Gel shift
Human Pol alpha Boosalis MS1987 KM 35uM Reaction: Nucleotide incorporation; Substrate: ATP analog; Technique: Gel shift
Human Pol alpha Boosalis MS1987 Vmax 12.9 /minute Reaction: Nucleotide incorporation; Substrate: dATP; Technique: Gel shift
Human Pol alpha Boosalis MS1987 Vmax 3.1 /minute Reaction: Nucleotide incorporation; Substrate: dGTP; Technique: Gel shift
Human Pol alpha Boosalis MS1987 Vmax 1.7 /minute Reaction: Nucleotide incorporation; Substrate: dTTP; Technique: Gel shift
Human Pol alpha Boosalis MS1987 Vmax 1.7 /minute Reaction: Nucleotide incorporation; Substrate: dCTP; Technique: Gel shift
Human Pol alpha Boosalis MS1987 Vmax 13 /minute Reaction: Nucleotide incorporation; Substrate: ATP analog; Technique: Gel shift

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