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

Nathan A. Tanner, Samir M. Hamdan, Slobodan Jergic, Patrick M. Schaeffer, Nicholas E. Dixon, Antoine M. Van Oijen*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

102 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)170-176
Number of pages7
JournalNature Structural and Molecular Biology
Volume15
Issue number2
DOIs
StatePublished - Feb 1 2008

ASJC Scopus subject areas

  • Structural Biology
  • Molecular Biology

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