Fidelity of Escherichia coli DNA polymerase III holoenzyme - The effects of beta,gamma complex processivity proteins and epsilon proofreading exonuclease on nucleotide misincorporation efficiencies

The fidelity of Escherichia coli DNA polymerase III (pol III), is measured and the effects of beta, gamma processivity and epsilon proofreading subunits are evaluated using a gel kinetic assay, Pol III holoenzyme synthesizes DNA with extremely high fidelity, misincorporating dTMP, dAMP, and dGMP opposite a template G target with efficiencies f(inc) = 5.6 x 10(-6), 4.2 x 10(-7) and 7 x 10(-7), respectively. Elevated dGMP.G and dTMP.G misincorporation efficiencies of 3.2 x 10(-5) and 5.8 x 10(-4), attributed to a ''dNTP-stabilized'' DNA misalignment mechanism, occur when C and A, respectively, are located one base downstream from the template target G. At least 92% of mis-inserted nucleotides are excised by pol III holoenzyme in the absence of a next correct ''rescue'' nucleotide, As rescue dNTP concentrations are increased, pol III holoenzyme suffers a maximum 8-fold reduction in fidelity as proofreading of mispaired primer termini are reduced in competition with incorporation of a next correct nucleotide, Compared with pol III holoenzyme, the alpha holoenzyme, which cannot proofread, has 47-, 32-, and 13-fold higher misincorporation rates for dGMP.G, dTMP.G, and dAMP.G mispairs. Both the beta, gamma complex and the downstream nucleotide have little effect on the fidelity of catalytic alpha subunit, An analysis of the gel kinetic fidelity assay when multiple polymerase-DNA encounters occur is presented in the ''Appendix'' (see Fygenson, D. K., and Goodman, M. F. (1997) J. Biol. Chem, 272, 27931-27935 (accompanying paper)).