Dynamics of Proofreading by the E. coli Pol III Replicase

Cell Chem Biol. 2018 Jan 18;25(1):57-66.e4. doi: 10.1016/j.chembiol.2017.09.008. Epub 2017 Nov 2.

Abstract

The αɛθ core of Escherichia coli DNA polymerase III (Pol III) associates with the β2 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 β2-associated Pol III core, and observed that both properties are enhanced by mutational strengthening of the interaction between ɛ and β2. Thus, the ɛ-β2 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 β2.

Keywords: Pol III replicase; proofreading; single-molecule flow-stretching; single-molecule fluorescence.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • DNA Polymerase III / metabolism*
  • Escherichia coli / enzymology*
  • Polymerization

Substances

  • DNA Polymerase III