Single-molecule studies of fork dynamics in Escherichia coli DNA replication

Journal Publication ResearchOnline@JCU
Tanner, Nathan A.;Hamdan, Samir M.;Jergic, Slobodan;Loscha, Karin V.;Schaeffer, Patrick M.;Dixon, Nicholas E.;van Oijen, Antoine M.
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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15

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1545-9985

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2

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Nature Publishing Group

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DOI

10.1038/nsmb0908-998a