Abstract
DNA synthesis, carried out by DNA polymerases, requires balancing speed and accuracy for faithful replication of the genome. High fidelity DNA polymerases contain a 3′–5′ exonuclease domain that can remove misincorporated nucleotides on the 3′ end of the primer strand, a process called proofreading. The E. coli replicative polymerase, DNA polymerase III, has spatially separated (~55 Å apart) polymerase and exonuclease subunits. Here, we report on the dynamics of E. coli DNA polymerase III proofreading in the presence of its processivity factor, the β2-sliding clamp, at varying base pair termini using single-molecule FRET. We find that the binding kinetics do not depend on the base identity at the termini, indicating a tolerance for DNA mismatches. Further, our single-molecule data and MD simulations show two previously unobserved features: (1) DNA Polymerase III is a highly dynamic protein that adopts multiple conformational states while bound to DNA with matched or mismatched ends, and (2) an exonuclease-deficient DNA polymerase III has reduced conformational flexibility. Overall, our single-molecule experiments provide high time-resolution insight into a mechanism that ensures high fidelity DNA replication to maintain genome integrity.
Highlights
DNA synthesis, carried out by DNA polymerases, requires balancing speed and accuracy for faithful replication of the genome
The single-molecule Forster resonance energy transfer (smFRET) assay enables us to monitor proofreading dynamics and kinetics of Pol III core binding on DNA at matched and mismatched termini (Fig. 1)
DNA was labeled with a FRET donor (Cy3) and the Pol III core with an acceptor (Cy5) on the y subunit to monitor conformational dynamics upon protein– DNA binding (Fig. 1A)
Summary
The DNA polymerase III holoenzyme is the replicative DNA polymerase in E. coli comprising ten proteins with a total mass of B10 MDa. The exonuclease binds the accessory protein y to form the trimeric complex Pol III core (i.e., a–e–y).[12,13] The polymerase and exonuclease active sites are separated by B55 Å.14. How these two subunits work together to coordinate proofreading while balancing fast and accurate DNA synthesis remains largely unknown. G:AA 50 CATAATATCC TCAGGAGTCC TTCGTCCTAG TACTACTCA 30 AÀACAGG AAGCAGGATC ATGATGAGT 50 a Template (top) and primer (bottom) strands. We tested Pol III core dynamics at the site of a double mismatch (G:AA, DNA sequence, Table 1). Our data reveal perturbations in proofreading dynamics at both the DNA and protein level, most notably regarding structural changes at the DNA terminus and with the Exo-deficient Pol III mutant
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