Letter | Published:

Replisome speed determines the efficiency of the Tus−Ter replication termination barrier

Nature volume 525, pages 394398 (17 September 2015) | Download Citation

Abstract

In all domains of life, DNA synthesis occurs bidirectionally from replication origins. Despite variable rates of replication fork progression, fork convergence often occurs at specific sites1. Escherichia coli sets a ‘replication fork trap’ that allows the first arriving fork to enter but not to leave the terminus region2,3,4,5. The trap is set by oppositely oriented Tus-bound Ter sites that block forks on approach from only one direction3,4,5,6,7. However, the efficiency of fork blockage by Tus–Ter does not exceed 50% in vivo despite its apparent ability to almost permanently arrest replication forks in vitro8,9. Here we use data from single-molecule DNA replication assays and structural studies to show that both polarity and fork-arrest efficiency are determined by a competition between rates of Tus displacement and rearrangement of Tus–Ter interactions that leads to blockage of slower moving replisomes by two distinct mechanisms. To our knowledge this is the first example where intrinsic differences in rates of individual replisomes have different biological outcomes.

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Accessions

Data deposits

Atomic coordinates and structure factors for the reported crystal structures have been deposited at the Protein Data Bank under accession codes 4XR0 (Tus–UGLT fork), 4XR1 (Tus–TGTA fork), 4XR2 (H144A–WT fork) and 4XR3 (Tus-UGLC).

References

  1. 1.

    et al. Random and site-specific replication termination. Methods Mol. Biol. 521, 35–53 (2009)

  2. 2.

    , & The terminus region of the Escherichia coli chromosome contains two separate loci that exhibit polar inhibition of replication. Proc. Natl Acad. Sci. USA 84, 1754–1758 (1987)

  3. 3.

    & Sequence-specific interactions in the Tus-Ter complex and the effect of base pair substitutions on arrest of DNA replication in Escherichia coli. J. Biol. Chem. 272, 26448–26456 (1997)

  4. 4.

    , , & Replication termination in Escherichia coli: structure and antihelicase activity of the Tus-Ter complex. Microbiol. Mol. Biol. Rev. 69, 501–526 (2005)

  5. 5.

    , , & The replication fork trap and termination of chromosome replication. Mol. Microbiol. 70, 1323–1333 (2008)

  6. 6.

    et al. A molecular mousetrap determines polarity of termination of DNA replication in E. coli. Cell 125, 1309–1319 (2006)

  7. 7.

    & Mechanisms of polar arrest of a replication fork. Mol. Microbiol. 72, 279–285 (2009)

  8. 8.

    , & Tus-mediated arrest of DNA replication in Escherichia coli is modulated by DNA supercoiling. Mol. Microbiol. 58, 758–773 (2005)

  9. 9.

    & Termination structures in the Escherichia coli chromosome replication fork trap. J. Mol. Biol. 387, 532–539 (2009)

  10. 10.

    , , & Biophysical characteristics of Tus, the replication arrest protein of Escherichia coli. J. Biol. Chem. 269, 4027–4034 (1994)

  11. 11.

    , , , & Structure of a replication-terminator protein complexed with DNA. Nature 383, 598–603 (1996)

  12. 12.

    et al. DNA primase acts as a molecular brake in DNA replication. Nature 439, 621–624 (2006)

  13. 13.

    et al. Single-molecule studies of fork dynamics in Escherichia coli DNA replication. Nat. Struct. Mol. Biol. 15, 170–176 (2008)

  14. 14.

    et al. A direct proofreader-clamp interaction stabilizes the Pol III replicase in the polymerization mode. EMBO J. 32, 1322–1333 (2013)

  15. 15.

    et al. Real-time single-molecule observation of rolling-circle DNA replication. Nucleic Acids Res. 37, e27 (2009)

  16. 16.

    , , , & Dynamics of DNA replication loops reveal temporal control of lagging-strand synthesis. Nature 457, 336–339 (2009)

  17. 17.

    , , & Single-molecule analysis reveals that the lagging strand increases replisome processivity but slows replication fork progression. Proc. Natl Acad. Sci. USA 106, 13236–13241 (2009)

  18. 18.

    et al. A single-molecule approach to DNA replication in Escherichia coli cells demonstrated that DNA polymerase III is a major determinant of fork speed. Mol. Microbiol. 90, 584–596 (2013)

  19. 19.

    & Differential Tus-Ter binding and lock formation: implications for DNA replication termination in Escherichia coli. Mol. Biosyst. 8, 2783–2791 (2012)

  20. 20.

    et al. Interaction of the Escherichia coli replication terminator protein (Tus) with DNA: a model derived from DNA-binding studies of mutant proteins by surface plasmon resonance. Biochemistry 39, 11989–11999 (2000)

  21. 21.

    et al. Replication termination mechanism as revealed by Tus-mediated polar arrest of a sliding helicase. Proc. Natl Acad. Sci. USA 105, 12831–12836 (2008)

  22. 22.

    , , & DnaB helicase activity is modulated by DNA geometry and force. Biophys. J. 99, 2170–2179 (2010)

  23. 23.

    , , & Coupling of a replicative polymerase and helicase: a τ-DnaB interaction mediates rapid replication fork movement. Cell 84, 643–650 (1996)

  24. 24.

    & Molecular traffic jams on DNA. Annu. Rev. Biophys. 42, 241–263 (2013)

  25. 25.

    et al. Flexibility revealed by the 1.85 Å crystal structure of the β sliding-clamp subunit of Escherichia coli DNA polymerase III. Acta Crystallogr. D 59, 1192–1199 (2003)

  26. 26.

    ϕX174-type primosomal proteins: purification and assay. Methods Enzymol. 262, 507–521 (1995)

  27. 27.

    et al. Blu-Ice and the Distributed Control System: software for data acquisition and instrument control at macromolecular crystallography beamlines. J. Synchrotron Radiat. 9, 401–406 (2002)

  28. 28.

    & Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326 (1997)

  29. 29.

    & MOLREP: an automated program for molecular replacement. J. Appl. Crystallogr. 30, 1022–1025 (1997)

  30. 30.

    , & Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr. D 53, 240–255 (1997)

  31. 31.

    , & Use of TLS parameters to model anisotropic displacements in macromolecular refinement. Acta Crystallogr. D 57, 122–133 (2001)

  32. 32.

    , , & Features and development of Coot. Acta Crystallogr. D 66, 486–501 (2010)

  33. 33.

    The PyMOL Molecular Graphics System, Version 1.5.0.4, Schrödinger, LLC

  34. 34.

    et al. Two mechanisms coordinate replication termination by the Escherichia coli Tus-Ter complex. Nucleic Acids Res. 43, 5924–5935 (2015)

  35. 35.

    et al. Single-molecule kinetics of lambda exonuclease reveal base dependence and dynamic disorder. Science 301, 1235–1238 (2003)

  36. 36.

    , , & Entropic elasticity of lambda-phage DNA. Science 265, 1599–1600 (1994)

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Acknowledgements

We thank A. van Oijen for critical comments and the groups of N. Dekker and S. Patel for helpful discussions. This research was supported by the King Abdullah University of Science and Technology through core funding (to S.M.H.) and a Faculty Initiated Collaborative Award (to S.M.H. and N.E.D.), and by the Australian Research Council (DP0877658 to N.E.D. and A.J.O.; DP0984797 to N.E.D.), including an Australian Professorial Fellowship to N.E.D. and a Future Fellowship (FT0990287) to A.J.O. X-ray crystallographic data were collected at the Australian Synchrotron, Victoria, Australia.

Author information

Affiliations

  1. Division of Biological and Environmental Sciences and Engineering, King Abdullah University of Science and Technology, Thuwal 23955, Saudi Arabia

    • Mohamed M. Elshenawy
    • , Mohamed A. Sobhy
    • , Masateru Takahashi
    •  & Samir M. Hamdan
  2. Centre for Medical & Molecular Bioscience, Illawarra Health & Medical Research Institute and University of Wollongong, New South Wales 2522, Australia

    • Slobodan Jergic
    • , Zhi-Qiang Xu
    • , Aaron J. Oakley
    •  & Nicholas E. Dixon

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Contributions

M.M.E. designed and carried out the single-molecule replication assays; M.M.E., M.A.S. and M.T. established the single-molecule replication assays; S.J. designed and carried out SPR measurements; S.J. and Z.-Q.X. isolated proteins; Z.-Q.X. and A.J.O. crystallized complexes, collected X-ray data and refined crystal structures. M.M.E., S.J., N.E.D. and S.M.H. designed the research and wrote the article. All authors analysed the data, discussed the results and commented on the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Nicholas E. Dixon or Samir M. Hamdan.

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https://doi.org/10.1038/nature14866

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