Mike O'Donnell

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)).

Efficiency and accuracy of SOS-induced DNA polymerases replicating benzo[a]pyrene-7,8-diol 9,10-epoxide A and G adducts.

Nucleotide incorporation fidelity, mismatch extension, and translesion DNA synthesis efficiencies were determined using SOS-induced Escherichia coli DNA polymerases (pol) II, IV, and V to copy 10R and 10S isomers oftrans-opened benzo[a]pyrene-7,8-diol 9,10-epoxide (BaP DE) A and G adducts. A-BaP DE adducts were bypassed by pol V with moderate accuracy and considerably higher efficiency than by pol II or IV. Error-prone pol V copied G-BaP DE-adducted DNA poorly, forming A·G-BaP DE-S and -R mismatches over C·G-BaP DE-S and -R correct matches by factors of ∼350- and 130-fold, respectively, even favoring G·G-BaP DE mismatches over correct matches by factors of 2–4-fold. In contrast, pol IV bypassed G-BaP DE adducts with the highest efficiency and fidelity, making misincorporations with a frequency of 10−2 to 10−4 depending on sequence context. G-BaP DE-S-adducted M13 DNA yielded 4-fold fewer plaques when transfected into SOS-induced ΔdinB (pol IV-deficient) mutant cells compared with the isogenic wild-typeE. coli strain, consistent with the in vitrodata showing that pol IV was most effective by far at copying the G-BaP DE-S adduct. SOS polymerases are adept at copying a variety of lesions, but the relative contribution of each SOS polymerase to copying damaged DNA appears to be determined by the lesions identity.

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.

Fidelity of Eucaryotic DNA Polymerase δ Holoenzyme fromSchizosaccharomyces pombe

The fidelity of Schizosaccharomyces pombe DNA polymerase δ was measured in the presence or absence of its processivity subunits, proliferating cell nuclear antigen (PCNA) sliding clamp and replication factor C (RFC) clamp-loading complex, using a synthetic 30-mer primer/100-mer template. Synthesis by pol δ alone was distributive. Processive synthesis occurred in the presence of PCNA, RFC, and Escherichia coli single strand DNA-binding protein (SSB) and required the presence of ATP. “Passive” self-loading of PCNA onto DNA takes place in the absence of RFC, in an ATP-independent reaction, which was strongly inhibited by SSB. The nucleotide substitution error rate for pol δ holoenzyme (HE) (pol δ + PCNA + RFC) was 4.6 × 10−4 for T·G mispairs, 5.3 × 10−5 for G·G mispairs, and 4.5 × 10−6 for A·G mispairs. The T·G misincorporation frequency for pol δ without the accessory proteins was unchanged. The fidelity of pol δ HE was between 1 and 2 orders of magnitude lower than that measured for the E. coli pol III HE at the same template position. This relatively low fidelity was caused by inefficient proofreading by the S. pombepolymerase-associated proofreading exonuclease. The S. pombe 3′-exonuclease activity was also extremely inefficient in excising primer-3′-terminal mismatches in the absence of dNTP substrates and in hydrolyzing single-stranded DNA. A comparison of pol δ HE with E. coli pol IIIα HE (lacking the proofreading exonuclease subunit) showed that both holoenzymes exhibit similar error rates for each mispair.

How should we teach lumbar manipulation? A consensus study

BACKGROUND Spinal manipulation is an effective intervention for low back pain, yet there is little consistency in how this skill is taught. OBJECTIVES The purpose of this study was to identify what educators and clinicians believe are important characteristics of the patient and operator position prior to side-lying lumbar manipulation and the patient position and operator motion during the manipulative thrust. DESIGN A multi-disciplinary correspondence-based Delphi method. METHODS Three rounds of questionnaires were sent to physical therapists, osteopaths and chiropractors. Consensus was established in Round 3 if at least 75% of respondents identified a characteristic as very important/extremely important on a 5-point Likert scale. RESULTS 265 educators and clinicians completed the three rounds of questioning. There was consensus that localization to target segment, patient comfort, table height, and logrolling the patient towards the operator are important characteristics of patient position during the preparatory phase. During the manipulation phase, respondents agreed that it is important to maintain localization to the segment and rotate the patients pelvis and lumbar spine. For the operator characteristics, consensus was reached for the following items; moving up and over the patient, maintaining contact using forearms, and close contact between the operator and patient (preparatory phase); generating force through the body and legs, dropping the body downwards, maintaining localization, and providing a high-velocity and low-amplitude thrust (manipulation phase). CONCLUSIONS This Delphi study successfully identified key characteristics of patient position and operator position and motion for effective delivery of side-lying lumbar spine manipulations.