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

Dynamics of Proofreading by the E. coli Pol III Replicase.

Author information

1
Department of Physics, Pohang University of Science & Technology (POSTECH), Pohang 37673, Korea.
2
Centre for Medical and Molecular Bioscience, University of Wollongong & Illawarra Health and Medical Research Institute, Wollongong, NSW 2522, Australia.
3
Centre for Medical and Molecular Bioscience, University of Wollongong & Illawarra Health and Medical Research Institute, Wollongong, NSW 2522, Australia. Electronic address: nickd@uow.edu.au.
4
Department of Physics, Pohang University of Science & Technology (POSTECH), Pohang 37673, Korea; School of Interdisciplinary Bioscience and Bioengineering, POSTECH, Pohang 37673, Korea. Electronic address: jblee@postech.ac.kr.

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

PMID:
29104063
DOI:
10.1016/j.chembiol.2017.09.008
[Indexed for MEDLINE]
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