Fidelity of Escherichia coli DNA Polymerase III Holoenzyme

The fidelity of Escherichia coli DNA polymerase III (pol III) is measured and the effects of β, γ processivity and ε 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 × 10−6, 4.2 × 10−7, and 7 × 10−7, respectively. Elevated dGMP·G and dTMP·G misincorporation efficiencies of 3.2 × 10−5 and 5.8 × 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 misinserted 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 α holoenzyme, which cannot proofread, has 47-, 32-, and 13-fold higher misincorporation rates for dGMP·G, dTMP·G, and dAMP·G mispairs. Both the β, γ complex and the downstream nucleotide have little effect on the fidelity of catalytic α 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)). The fidelity of Escherichia coli DNA polymerase III (pol III) is measured and the effects of β, γ processivity and ε 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 × 10−6, 4.2 × 10−7, and 7 × 10−7, respectively. Elevated dGMP·G and dTMP·G misincorporation efficiencies of 3.2 × 10−5 and 5.8 × 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 misinserted 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 α holoenzyme, which cannot proofread, has 47-, 32-, and 13-fold higher misincorporation rates for dGMP·G, dTMP·G, and dAMP·G mispairs. Both the β, γ complex and the downstream nucleotide have little effect on the fidelity of catalytic α 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)). The first in vitro measurement of DNA synthesis fidelity was carried out by Kornberg and co-workers in 1962 (1Trautner T.A. Swartz M.N. Kornberg A. Proc. Natl. Acad. Sci. U. S. A. 1962; 48: 449-455Crossref PubMed Scopus (62) Google Scholar) to analyze the mutagenic behavior of 5-bromouracil. Pol I 1The abbreviations used are: pol I, E. coli DNA polymerase I; pol III, E. coli DNA polymerase III; pol III core, comprised of α (polymerase), ε (3′ → 5′ proofreading exonuclease), and θ; pol III holoenzyme, comprised of pol III core + β (sliding processivity clamp) + γ complex (clamp loading complex containing γ, γ′, δ, χ, ψ); α holoenzyme, comprised of α + β, γ complex; pol δ; eucaryotic DNA polymerase δ; p/t, primer/template; SSB, E. coli single-stranded DNA-binding protein; T (in boldface), refers to the template target site at which fidelity is measured and should not be confused with T which refers to a template thymine; SCH, single completed hit conditions referring to extension of a primer via interaction with a DNA polymerase at most once, followed by polymerase dissociation; MCH, multiple completed hit conditions referring to extension of a primer via multiple interactions with DNA polymerase. was found to misincorporate dGMP more readily opposite template bromouracil than opposite T, thus providing a biochemical basis for understanding to → in Escherichia coli and E. 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