The αɛθ core of Escherichia coli DNA polymerase III (Pol III) associates with the β sliding clamp to processively synthesize DNA and remove misincorporated nucleotides. The α subunit is the polymerase while ɛ is the 3' to 5' proofreading exonuclease. In contrast to the polymerase activity of Pol III, dynamic features of proofreading are poorly understood. We used single-molecule assays to determine the excision rate and processivity of the β-associated Pol III core, and observed that both properties are enhanced by mutational strengthening of the interaction between ɛ and β. Thus, the ɛ-β contact is maintained in both the synthesis and proofreading modes. Remarkably, single-molecule real-time fluorescence imaging revealed the dynamics of transfer of primer-template DNA between the polymerase and proofreading sites, showing that it does not involve breaking of the physical interaction between ɛ and β.
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