Single-Molecule Studies of Fork Dynamics in E. coli DNA Replication

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 kb, 8-fold higher than that of primer extension by Pol III alone. Addition of the primase DnaG causes a 3-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 2-3 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.