Single-Molecule Studies of Fork Dynamics of Escherichia coli DNA Replication

We present single-molecule studies of the replication machinery of Escherichia coli and describe the visualization of 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 to the replisome 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 the propagating DnaB destabilizes the replisome. The 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.