Abstract
A gel fidelity assay, previously used in the analysis of DNA polymerases having no associated 3' to 5' exonuclease activity, has been generalized for use with polymerases that contain exonucleolytic proofreading. The main purpose of this study was the development of a general analysis, using a standard Markov model, to convert experimentally observed DNA primer gel bands arising from insertion and proofreading of right and wrong deoxyribonucleotides, into nucleotide incorporation velocities and, most importantly, fidelities. The model has been applied primarily to an analysis of polymerase kinetics and fidelity in the presence of a next correct rescue dNTP, but the model can be conveniently modified to investigate other experimental designs. In the presence of rescue dNTP, direct competition occurs between excision or extension of a mismatch. At concentrations of rescue dNTP sufficient to suppress the gel band intensity at the mismatch target site, nucleotide incorporation and misincorporation rates can be obtained from the ratios of gel band intensities 3' (downstream) and 5' (upstream) to the target site, measured as a function dNTP concentration for "wrong" and "right" dNTP substrates. The polymerase misincorporation efficiency, in the presence of proofreading, is given by the ratio of wrong to right incorporation efficiencies, Vmax/Km, obtained from the gel band ratios. The bacteriophage T4 polymerase with a highly active 3'-exonuclease activity was used to illustrate the assay. Nucleotide misincorporation efficiencies measured at several template sites were dCMP.A approximately equal to 10(-6), dGMP.A approximately equal to 10(-5), dTMP.T approximately equal to 2 x 10(-4), and dAMP.A < 10(-7). Proofreading of the dGMP.A mispair was suppressed by about 3-fold in the presence of high concentrations of next correct "rescue" dNTP causing a concomitant reduction in the fidelity of dGMP.A to about 3 x 10(-5).
Highlights
IntroductionThe costs of publication of this article were defrayed in part by the payment of page charges
Measurements of the dependence of integrated gel band intensities as a function of the concentration of right and wrong dNTP substrates have been used to deduce the fidelity of nucleotide insertion for DNA polymerases devoid of 3'-exonucleolytic proofreading activity [13, 24, 25]
Our primary experimental objective in this paper is to investigate the use of a gel kinetic assay to measure DNA polymerase fidelity in the presence of proofreading
Summary
The costs of publication of this article were defrayed in part by the payment of page charges. This article must be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Perhaps involving subtle differences in the interactions linking polymerase, matched and mismatched dNTP substrates, and primer-template DNA, can contribute to enzyme-specific variations in mutational spectra and nonrandom error distributions. The availability of a rapid assay measuring fidelity at arbitrary template locations would be useful for analyzing how differences in polymerases and primer template sequences contribute to each individual type of base substitution and frameshift mutation. A "gel kinetic" assay, in which incorporation of matched and mismatched dNTP substrates are measured, in separate reactions, as a function ofdNTP concentration, has been used as an alternative method of deducing insertion fidelity for polymerases [2, 24, 25], In the gel kinetic assay, extension of 32p_
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