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

111 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 2008
Externally publishedYes

Bibliographical note

Funding Information:
The authors thank J. Loparo, Harvard Medical School, Boston, for construction of a flow cell heating apparatus and critical reading of the manuscript, and A.Y. Park and M. Mulcair, Australian National University, Canberra, for preparation of several proteins. We thank S. Moskowitz, Advanced Medical Graphics, for illustrations. This work was supported in part by funding from the US National Institutes of Health and National Science Foundation (A.M.v.O.) and by a grant from the Australian Research Council (N.E.D.).

ASJC Scopus subject areas

  • Structural Biology
  • Molecular Biology

Fingerprint

Dive into the research topics of 'Single-molecule studies of fork dynamics in Escherichia coli DNA replication'. Together they form a unique fingerprint.

Cite this