Abstract

We present single-molecule studies of the Escherichia coli replication machinery. We visualize individual E. coli DNA polymerase III (Pol III) holoenzymes engaging in primer extension and leading-strand synthesis. When coupled to the replicative helicase DnaB, Pol III mediates leading-strand synthesis with a processivity of 10.5 kilobases (kb), eight-fold higher than that by Pol III alone. Addition of the primase DnaG causes a three-fold reduction in the processivity of leading-strand synthesis, an effect dependent upon the DnaB-DnaG protein-protein interaction rather than primase activity. A single-molecule analysis of the replication kinetics with varying DnaG concentrations indicates that a cooperative binding of two or three DnaG monomers to DnaB halts synthesis. Modulation of DnaB helicase activity through the interaction with DnaG suggests a mechanism that prevents leading-strand synthesis from outpacing lagging-strand synthesis during slow primer synthesis on the lagging strand.

1. Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 250 Longwood Avenue, Boston, Massachusetts 02115, USA.
2. Graduate Program in Biological and Biomedical Sciences, Harvard Medical School, 250 Longwood Avenue, Boston, Massachusetts 02115, USA.
3. Research School of Chemistry, Australian National University, Canberra, Australian Capital Territory 0200, Australia.
4. School of Chemistry, Building 18, Northfields Avenue, University of Wollongong, New South Wales 2522, Australia.
5. School of Pharmacy and Molecular Sciences, James Cook University, Townsville, Queensland 4811, Australia.

Correspondence to: Antoine M van Oijen¹ e-mail: antoine_van_oijen@hms.harvard.edu

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