Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

A solution to release twisted DNA during chromosome replication by coupled DNA polymerases

Abstract

Chromosomal replication machines contain coupled DNA polymerases that simultaneously replicate the leading and lagging strands1. However, coupled replication presents a largely unrecognized topological problem. Because DNA polymerase must travel a helical path during synthesis, the physical connection between leading- and lagging-strand polymerases causes the daughter strands to entwine, or produces extensive build-up of negative supercoils in the newly synthesized DNA2,3,4. How DNA polymerases maintain their connection during coupled replication despite these topological challenges is unknown. Here we examine the dynamics of the Escherichia coli replisome, using ensemble and single-molecule methods, and show that the replisome may solve the topological problem independent of topoisomerases. We find that the lagging-strand polymerase frequently releases from an Okazaki fragment before completion, leaving single-strand gaps behind. Dissociation of the polymerase does not result in loss from the replisome because of its contact with the leading-strand polymerase. This behaviour, referred to as ‘signal release’, had been thought to require a protein, possibly primase, to pry polymerase from incompletely extended DNA fragments5,6,7. However, we observe that signal release is independent of primase and does not seem to require a protein trigger at all. Instead, the lagging-strand polymerase is simply less processive in the context of a replisome. Interestingly, when the lagging-strand polymerase is supplied with primed DNA in trans, uncoupling it from the fork, high processivity is restored. Hence, we propose that coupled polymerases introduce topological changes, possibly by accumulation of superhelical tension in the newly synthesized DNA, that cause lower processivity and transient lagging-strand polymerase dissociation from DNA.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Figure 1: The topological problem caused by coupled leading- and lagging-strand polymerases.
Figure 2: Signal release does not require primase and correlates with increasing Okazaki fragment length.
Figure 3: Lagging-strand polymerase processivity is restored using a separate DNA molecule.
Figure 4: Single-molecule total internal reflection fluorescence microscopy.

References

  1. Kornberg, A. & Baker, T. A. DNA Replication 2nd edn (W. H. Freeman, 1992)

    Google Scholar 

  2. Ullsperger, C. J., Volgodskii, A. V. & Cozzarelli, N. R. Unlinking of DNA by Topoisomerases during DNA Replication Vol. 9 (Springer, 1995)

    Book  Google Scholar 

  3. Hingorani, M. M. & O’Donnell, M. Sliding clamps: a (tail)ored fit. Curr. Biol. 10, R25–R29 (2000)

    Article  CAS  Google Scholar 

  4. Wyman, C. & Botchan, M. DNA replication. A familiar ring to DNA polymerase processivity. Curr. Biol. 5, 334–337 (1995)

    Article  CAS  Google Scholar 

  5. Hamdan, S. M., Loparo, J. J., Takahashi, M., Richardson, C. C. & van Oijen, A. M. Dynamics of DNA replication loops reveal temporal control of lagging-strand synthesis. Nature 457, 336–339 (2009)

    Article  ADS  CAS  Google Scholar 

  6. Li, X. & Marians, K. J. Two distinct triggers for cycling of the lagging strand polymerase at the replication fork. J. Biol. Chem. 275, 34757–34765 (2000)

    Article  CAS  Google Scholar 

  7. Yang, J., Nelson, S. W. & Benkovic, S. J. The control mechanism for lagging strand polymerase recycling during bacteriophage T4 DNA replication. Mol. Cell 21, 153–164 (2006)

    Article  CAS  Google Scholar 

  8. Johnson, A. & O’Donnell, M. Cellular DNA replicases: components and dynamics at the replication fork. Annu. Rev. Biochem. 74, 283–315 (2005)

    Article  CAS  Google Scholar 

  9. Postow, L., Peter, B. J. & Cozzarelli, N. R. Knot what we thought before: the twisted story of replication. Bioessays 21, 805–808 (1999)

    Article  CAS  Google Scholar 

  10. Chastain, P. D., II, Makhov, A. M., Nossal, N. G. & Griffith, J. Architecture of the replication complex and DNA loops at the fork generated by the bacteriophage t4 proteins. J. Biol. Chem. 278, 21276–21285 (2003)

    Article  CAS  Google Scholar 

  11. Vologodskii, A. V., Lukashin, A. V., Anshelevich, V. V. & Frank-Kamenetskii, M. D. Fluctuations in superhelical DNA. Nucleic Acids Res. 6, 967–982 (1979)

    Article  CAS  Google Scholar 

  12. Leu, F. P., Georgescu, R. & O’Donnell, M. Mechanism of the E. coli tau processivity switch during lagging-strand synthesis. Mol. Cell 11, 315–327 (2003)

    Article  CAS  Google Scholar 

  13. Espeli, O. & Marians, K. J. Untangling intracellular DNA topology. Mol. Microbiol. 52, 925–931 (2004)

    Article  CAS  Google Scholar 

  14. Stockum, A., Lloyd, R. G. & Rudolph, C. J. On the viability of Escherichia coli cells lacking DNA topoisomerase I. BMC Microbiol. 12, 26 (2012)

    Article  CAS  Google Scholar 

  15. Stupina, V. A. & Wang, J. C. Viability of Escherichia coli topA mutants lacking DNA topoisomerase I. J. Biol. Chem. 280, 355–360 (2005)

    Article  CAS  Google Scholar 

  16. Yao, N. Y., Georgescu, R. E., Finkelstein, J. & O’Donnell, M. E. 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)

    Article  ADS  CAS  Google Scholar 

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

    Article  Google Scholar 

  18. O’Donnell, M. E. & Kornberg, A. Complete replication of templates by Escherichia coli DNA polymerase III holoenzyme. J. Biol. Chem. 260, 12884–12889 (1985)

    PubMed  Google Scholar 

  19. McHenry, C. S. DNA replicases from a bacterial perspective. Annu. Rev. Biochem. 80, 403–436 (2011)

    Article  CAS  Google Scholar 

  20. Wu, C. A., Zechner, E. L., Reems, J. A., McHenry, C. S. & Marians, K. J. Coordinated leading- and lagging-strand synthesis at the Escherichia coli DNA replication fork. V. Primase action regulates the cycle of Okazaki fragment synthesis. J. Biol. Chem. 267, 4074–4083 (1992)

    CAS  PubMed  Google Scholar 

  21. Marko, J. F. & Siggia, E. D. Stretching DNA. Macromolecules 28, 8759–8770 (1995)

    Article  ADS  CAS  Google Scholar 

  22. Dorman, C. J. DNA supercoiling and bacterial gene expression. Sci. Prog. 89, 151–166 (2006)

    Article  CAS  Google Scholar 

  23. Travers, A. & Muskhelishvili, G. A common topology for bacterial and eukaryotic transcription initiation? EMBO Rep. 8, 147–151 (2007)

    Article  CAS  Google Scholar 

  24. Benyajati, C. & Worcel, A. Isolation, characterization, and structure of the folded interphase genome of Drosophila melanogaster. Cell 9, 393–407 (1976)

    Article  CAS  Google Scholar 

  25. McInerney, P. & O’Donnell, M. Functional uncoupling of twin polymerases: mechanism of polymerase dissociation from a lagging-strand block. J. Biol. Chem. 279, 21543–21551 (2004)

    Article  CAS  Google Scholar 

  26. Yao, N., Hurwitz, J. & O’Donnell, M. Dynamics of beta and proliferating cell nuclear antigen sliding clamps in traversing DNA secondary structure. J. Biol. Chem. 275, 1421–1432 (2000)

    Article  CAS  Google Scholar 

  27. Onrust, R., Finkelstein, J., Turner, J., Naktinis, V. & O’Donnell, M. Assembly of a chromosomal replication machine: two DNA polymerases, a clamp loader, and sliding clamps in one holoenzyme particle. III. Interface between two polymerases and the clamp loader. J. Biol. Chem. 270, 13366–13377 (1995)

    Article  CAS  Google Scholar 

  28. Bailey, S., Wing, R. A. & Steitz, T. A. The structure of T. aquaticus DNA polymerase III is distinct from eukaryotic replicative DNA polymerases. Cell 126, 893–904 (2006)

    Article  CAS  Google Scholar 

  29. Georgescu, R. E. et al. Mechanism of polymerase collision release from sliding clamps on the lagging strand. EMBO J. 28, 2981–2991 (2009)

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank L. Langston, A. Libchaber and B. Michel for suggestions on the manuscript. We are also grateful to the National Institutes of Health (GM 38839) for supporting this work.

Author information

Authors and Affiliations

Authors

Contributions

I.K. and M.O.D. conceived the project, I.K. and R.G. performed experiments, and I.K., R.G. and M.O.D. designed the experiments, analysed data and wrote the paper.

Corresponding author

Correspondence to Mike E. O'Donnell.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Figures

This file contains Supplementary Figures 1-9. (PDF 2349 kb)

PowerPoint slides

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Kurth, I., Georgescu, R. & O'Donnell, M. A solution to release twisted DNA during chromosome replication by coupled DNA polymerases. Nature 496, 119–122 (2013). https://doi.org/10.1038/nature11988

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature11988

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing