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

Nature Structural & Molecular Biology volume 15, pages 170–176 (2008)Cite this article

A Corrigendum to this article was published on 01 September 2008

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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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Figure 1: Schematic representation of the E. coli replisome mediating coordinated DNA synthesis.
Figure 2: Single-molecule experimental setup.
Figure 3: Primer extension by the DNA polymerase III holoenzyme.
Figure 4: Leading-strand synthesis by Pol III holoenzyme coupled with DnaB helicase.
Figure 5: Replicative abortion is independent of DnaG primase activity.
Figure 6: Cooperative DnaG-DnaB interaction is dependent upon the RNA polymerase domain of primase.

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  • 18 June 2008

    In the version of this article initially published, Karin V. Loscha was missing from the list of authors. The error has been corrected in the HTML and PDF versions of the article.

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Acknowledgements

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.).

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Authors and Affiliations

  1. Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 250 Longwood Avenue, Boston, 02115, Massachusetts, USA

    Nathan A Tanner, Samir M Hamdan & Antoine M van Oijen

  2. Graduate Program in Biological and Biomedical Sciences, Harvard Medical School, 250 Longwood Avenue, Boston, 02115, Massachusetts, USA

    Nathan A Tanner

  3. Research School of Chemistry, Australian National University, Canberra, Australian Capital Territory 0200, Australia.,

    Slobodan Jergic, Karin V Loscha, Patrick M Schaeffer & Nicholas E Dixon

  4. School of Chemistry, Building 18, Northfields Avenue, University of Wollongong, New South Wales, 2522, Australia

    Slobodan Jergic & Nicholas E Dixon

  5. School of Pharmacy and Molecular Sciences, James Cook University, Townsville, 4811, Queensland, Australia

    Patrick M Schaeffer

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Contributions

N.A.T. performed single-molecule experiments, interpreted the data and wrote the manuscript; S.M.H. interpreted the data and designed experiments; S.J. and P.M.S. purified all proteins and complexes; N.E.D. designed experiments and wrote the manuscript; A.M.v.O. designed experiments, interpreted data and wrote the manuscript.

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Correspondence to Antoine M van Oijen.

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Tanner, N., Hamdan, S., Jergic, S. et al. Single-molecule studies of fork dynamics in Escherichia coli DNA replication. Nat Struct Mol Biol 15, 170–176 (2008). https://doi.org/10.1038/nsmb.1381

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