Dynamics of Proofreading by the E. coli Pol III Replicase

Abstract

The alpha epsilon theta core of Escherichia coli DNA polymerase III (Pol III) associates with the beta(2) sliding clamp to processively synthesize DNA and remove misincorporated nucleotides. The alpha subunit is the polymerase while epsilon 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 beta(2)-associated Pol III core, and observed that both properties are enhanced by mutational strengthening of the interaction between epsilon and beta(2). Thus, the epsilon-beta(2) 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 epsilon and beta(2) .