Slobodan Jergic¹, Nicholas P Horan¹, Mohamed M Elshenawy², Claire E Mason¹, Thitima Urathamakul¹, Kiyoshi Ozawa^1,3, Andrew Robinson^1,4, Joris M H Goudsmits⁵, Yao Wang¹, Xuefeng Pan^1,6, Jennifer L Beck¹, Antoine M van Oijen^4,5, Thomas Huber³, Samir M Hamdan² and Nicholas E Dixon¹

1. School of Chemistry, University of Wollongong, Wollongong, New South Wales, Australia
2. Division of Biological and Environmental Sciences and Engineering, King Abdullah University of Science and Technology, Thuwal, Saudi Arabia
3. Research School of Chemistry, Australian National University, Canberra, Australian Capital Territory, Australia
4. Zernike Institute for Advanced Materials, Groningen, The Netherlands
5. Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, USA

Correspondence to:

Nicholas E Dixon, School of Chemistry, University of Wollongong, Northfields Avenue, Wollongong, New South Wales 2522, Australia. Tel.:+61 2 42214346; Fax:+61 2 42214287; E-mail: nickd@uow.edu.au

⁶Permanent address: School of Life Science, Beijing Institute of Technology, Beijing 100081, China.

Received 26 October 2012; Accepted 7 December 2012

Processive DNA synthesis by the αεθ core of the Escherichia coli Pol III replicase requires it to be bound to the β₂ clamp via a site in the α polymerase subunit. How the ε proofreading exonuclease subunit influences DNA synthesis by α was not previously understood. In this work, bulk assays of DNA replication were used to uncover a non-proofreading activity of ε. Combination of mutagenesis with biophysical studies and single-molecule leading-strand replication assays traced this activity to a novel β-binding site in ε that, in conjunction with the site in α, maintains a closed state of the αεθ–β₂ replicase in the polymerization mode of DNA synthesis. The ε–β interaction, selected during evolution to be weak and thus suited for transient disruption to enable access of alternate polymerases and other clamp binding proteins, therefore makes an important contribution to the network of protein–protein interactions that finely tune stability of the replicase on the DNA template in its various conformational states.

• Keywords:
  • beta sliding clamp;
  • DNA polymerase III;
  • DNA replication;
  • Escherichia coli;
  • proofreading exonuclease