Linda B. Bloom

Fidelity of Escherichia coli DNA Polymerase III Holoenzyme THE EFFECTS OF β, γ COMPLEX PROCESSIVITY PROTEINS AND ε PROOFREADING EXONUCLEASE ON NUCLEOTIDE MISINCORPORATION EFFICIENCIES

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)).

Dynamics of loading the beta sliding clamp of DNA polymerase III onto DNA.

A “minimal” DNA primer-template system, consisting of an 80-mer template and 30-mer primer, supports processive DNA synthesis by DNA polymerase III core in the presence of the β sliding clamp, γ complex clamp loader, and single-stranded binding protein from Escherichia coli. This primer-template system was used to measure the loading of the β sliding clamp by the γ complex in an ATP-dependent reaction. Bound protein-DNA complexes were detected by monitoring fluorescence depolarization of DNA. Steady state and time-resolved anisotropies were measured, and stopped-flow pre-steady state fluorescence measurements allowed visualization of the loading reactions in real time. The rate of loading β onto DNA was 12 s−1, demonstrating that clamp assembly is rapid on the time scale required for lagging strand Okazaki fragment synthesis. The association rate appears to be limited by an intramolecular step occurring prior to the clamp-loading reaction, possibly the opening of the toroidal β dimer.

A Model for Escherichia coli DNA Polymerase III Holoenzyme Assembly at Primer/Template Ends DNA TRIGGERS A CHANGE IN BINDING SPECIFICITY OF THE γ COMPLEX CLAMP LOADER

The γ complex of the Escherichia coli DNA polymerase III holoenzyme assembles the β sliding clamp onto DNA in an ATP hydrolysis-driven reaction. Interactions between γ complex and primer/template DNA are investigated using fluorescence depolarization to measure binding of γ complex to different DNA substrates under steady-state and presteady-state conditions. Surprisingly, γ complex has a much higher affinity for single-stranded DNA (K d in the nmrange) than for a primed template (K d in the μm range) under steady-state conditions. However, when examined on a millisecond time scale, we find that γ complex initially binds very rapidly and with high affinity to primer/template DNA but is converted subsequently to a much lower affinity DNA binding state. Presteady-state data reveals an effective dissociation constant of 1.5 nm for the initial binding of γ complex to DNA and a dissociation constant of 5.7 μm for the low affinity DNA binding state. Experiments using nonhydrolyzable ATPγS show that ATP binding converts γ complex from a low affinity “inactive” to high affinity “active” DNA binding state while ATP hydrolysis has the reverse effect, thus allowing cycling between active and inactive DNA binding forms at steady-state. We propose that a DNA-triggered switch between active and inactive states of γ complex provides a two-tiered mechanism enabling γ complex to recognize primed template sites and load β, while preventing γ complex from competing with DNA polymerase III core for binding a newly loaded β·DNA complex.

Pre-steady State Analysis of the Assembly of Wild Type and Mutant Circular Clamps of Escherichia coli DNA Polymerase III onto DNA

The β protein, a dimeric ring-shaped clamp essential for processive DNA replication by Escherichia coli DNA polymerase III holoenzyme, is assembled onto DNA by the γ complex. This study examines the clamp loading pathway in real time, using pre-steady state fluorescent depolarization measurements to investigate the loading reaction and ATP requirements for the assembly of β onto DNA. Two β dimer interface mutants, L273A and L108A, and a nonhydrolyzable ATP analog, adenosine 5′-O-(3-thiotriphosphate) (ATPγS), have been used to show that ATP binding is required for γ complex and β to associate with DNA, but that a γ complex-catalyzed ATP hydrolysis is required for γ complex to release the β·DNA complex and complete the reaction. In the presence of ATP and γ complex, the β mutants associate with DNA as efficiently as wild type β. However, completion of the reaction is much slower with the β mutants because of decreased ATP hydrolysis by the γ complex, resulting in a much slower release of the mutants onto DNA. The effects of mutations in the dimer interface were similar to the effects of replacing ATP with ATPγS in reactions using wild type β. Thus, the assembly of β around DNA is coupled tightly to the ATPase activity of the γ complex, and completion of the assembly process requires ATP hydrolysis for turnover of the catalytic clamp loader.

The proofreading pathway of bacteriophage T4 DNA polymerase.

The base analog, 2-aminopurine (2AP), was used as a fluorescent reporter of the biochemical steps in the proofreading pathway catalyzed by bacteriophage T4 DNA polymerase. “Mutator” DNA polymerases that are defective in different steps in the exonucleolytic proofreading pathway were studied so that transient changes in fluorescence intensity could be equated with specific reaction steps. The G255S- and D131N-DNA polymerases can hydrolyze DNA, the final step in the proofreading pathway, but the mutator phenotype indicates a defect in one or more steps that prepare the primer-terminus for the cleavage reaction. The hydrolysis-defective D112A/E114A-DNA polymerase was also examined. Fluorescent enzyme-DNA complexes werepreformed in the absence of Mg2+, and then rapid mixing, stopped-flow techniques were used to determine the fate of the fluorescent complexes upon the addition of Mg2+. Comparisons of fluorescence intensity changes between the wild type and mutant DNA polymerases were used to model the exonucleolytic proofreading pathway. These studies are consistent with a proofreading pathway in which the protein loop structure that contains residue Gly255 functions in strand separation and transfer of the primer strand from the polymerase active center to form a preexonuclease complex. Residue Asp131 acts at a later step in formation of the preexonuclease complex.