Tanner, Nathan A., Hamdan, Samir M., Jergic, Slobodan, Loscha, Karin V., Schaeffer, Patrick M., Dixon, Nicholas E., and van Oijen, Antoine M. (2008) Single-molecule studies of fork dynamics in Escherichia coli DNA replication. Nature Structural and Molecular Biology, 15 (2). pp. 170-176.

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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.

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