Skip to main content

Thank you for visiting 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.

Dynamics of DNA replication loops reveal temporal control of lagging-strand synthesis


In all organisms, the protein machinery responsible for the replication of DNA, the replisome, is faced with a directionality problem. The antiparallel nature of duplex DNA permits the leading-strand polymerase to advance in a continuous fashion, but forces the lagging-strand polymerase to synthesize in the opposite direction. By extending RNA primers, the lagging-strand polymerase restarts at short intervals and produces Okazaki fragments1,2. At least in prokaryotic systems, this directionality problem is solved by the formation of a loop in the lagging strand of the replication fork to reorient the lagging-strand DNA polymerase so that it advances in parallel with the leading-strand polymerase. The replication loop grows and shrinks during each cycle of Okazaki fragment synthesis3. Here we use single-molecule techniques to visualize, in real time, the formation and release of replication loops by individual replisomes of bacteriophage T7 supporting coordinated DNA replication. Analysis of the distributions of loop sizes and lag times between loops reveals that initiation of primer synthesis and the completion of an Okazaki fragment each serve as a trigger for loop release. The presence of two triggers may represent a fail-safe mechanism ensuring the timely reset of the replisome after the synthesis of every Okazaki fragment.

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

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Get just this article for as long as you need it


Prices may be subject to local taxes which are calculated during checkout

Figure 1: Observation of replication loops.
Figure 2: Fluorescence imaging of coordinated DNA replication.
Figure 3: Replication loop dynamics depend on primase activity.
Figure 4: Signalling and collision mechanisms both serve to trigger loop release.


  1. Benkovic, S. J., Valentine, A. M. & Salinas, F. Replisome-mediated DNA replication. Annu. Rev. Biochem. 70, 181–208 (2001)

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  3. Alberts, B. M. et al. Studies on DNA replication in the bacteriophage T4 in vitro system. Cold Spring Harb. Symp. Quant. Biol. 47, 655–668 (1983)

    Article  PubMed  Google Scholar 

  4. Chastain, P. D., Makhov, A. M., Nossal, N. G. & Griffith, J. D. Analysis of the Okazaki fragment distributions along single long DNAs replicated by the bacteriophage T4 proteins. Mol. Cell 6, 803–814 (2000)

    Article  CAS  PubMed  Google Scholar 

  5. Park, K., Debyser, Z., Tabor, S., Richardson, C. C. & Griffith, J. D. Formation of a DNA loop at the replication fork generated by bacteriophage T7 replication proteins. J. Biol. Chem. 273, 5260–5270 (1998)

    Article  CAS  PubMed  Google Scholar 

  6. Carver, T. E., Sexton, D. J. & Benkovic, S. J. Dissociation of bacteriophage T4 DNA polymerase and its processivity clamp after completion of Okazaki fragment synthesis. Biochemistry 36, 14409–14417 (1997)

    Article  CAS  PubMed  Google Scholar 

  7. López de Saro, F. J., Georgescu, R. E. & O’Donnell, M. A peptide switch regulates DNA polymerase processivity. Proc. Natl Acad. Sci. USA 100, 14689–14694 (2003)

    Article  ADS  PubMed  PubMed Central  Google Scholar 

  8. Hacker, K. J. & Alberts, B. M. The rapid dissociation of the T4 DNA-polymerase holoenzyme when stopped by a DNA hairpin helix: a model for polymerase release following the termination of each Okazaki fragment. J. Biol. Chem. 269, 24221–24228 (1994)

    CAS  PubMed  Google Scholar 

  9. 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  PubMed  Google Scholar 

  10. 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  PubMed  Google Scholar 

  11. Nossal, N. G., Makhov, A. M., Chastain, P. D., Jones, C. E. & Griffith, J. D. Architecture of the bacteriophage T4 replication complex revealed with nanoscale biopointers. J. Biol. Chem. 282, 1098–1108 (2007)

    Article  CAS  PubMed  Google Scholar 

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

    CAS  PubMed  Google Scholar 

  13. Lee, J., Chastain, P. D., Griffith, J. D. & Richardson, C. C. Lagging strand synthesis in coordinated DNA synthesis by bacteriophage T7 replication proteins. J. Mol. Biol. 316, 19–34 (2002)

    Article  CAS  PubMed  Google Scholar 

  14. Tougu, K. & Marians, K. J. The interaction between helicase and primase sets the replication fork clock. J. Biol. Chem. 271, 21398–21405 (1996)

    Article  CAS  PubMed  Google Scholar 

  15. Richardson, C. C. Bacteriophage T7: minimal requirements for the replication of a duplex DNA molecule. Cell 33, 315–317 (1983)

    Article  CAS  PubMed  Google Scholar 

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

    Article  ADS  CAS  PubMed  Google Scholar 

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

    Article  CAS  Google Scholar 

  18. Hamdan, S. M. et al. Dynamic DNA helicase-DNA polymerase interactions assure processive replication fork movement. Mol. Cell 27, 539–549 (2007)

    Article  CAS  PubMed  Google Scholar 

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

    Article  ADS  CAS  PubMed  Google Scholar 

  20. Lee, J., Chastain, P. D., Kusakabe, T., Griffith, J. D. & Richardson, C. C. Coordinated leading and lagging strand DNA synthesis on a minicircular template. Mol. Cell 1, 1001–1010 (1998)

    Article  CAS  PubMed  Google Scholar 

  21. Frick, D. N. & Richardson, C. C. DNA primases. Annu. Rev. Biochem. 70, 39–80 (2001)

    Article  CAS  PubMed  Google Scholar 

  22. Qimron, U., Lee, S. J., Hamdan, S. M. & Richardson, C. C. Primer initiation and extension by T7 DNA primase. EMBO J. 25, 2199–2208 (2006)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Kusakabe, T. & Richardson, C. C. Gene 4 DNA primase of bacteriophage T7 mediates the annealing and extension of ribo-oligonucleotides at primase recognition sites. J. Biol. Chem. 272, 12446–12453 (1997)

    Article  CAS  PubMed  Google Scholar 

  24. Frick, D. N., Kumar, S. & Richardson, C. C. Interaction of ribonucleoside triphosphates with the gene 4 primase of bacteriophage T7. J. Biol. Chem. 274, 35899–35907 (1999)

    Article  CAS  PubMed  Google Scholar 

  25. Notarnicola, S. M., Mulcahy, H. L., Lee, J. & Richardson, C. C. The acidic carboxyl terminus of the bacteriophage T7 gene 4 helicase/primase interacts with T7 DNA polymerase. J. Biol. Chem. 272, 18425–18433 (1997)

    Article  CAS  PubMed  Google Scholar 

  26. Tabor, S., Huber, H. E. & Richardson, C. C. Escherichia coli thioredoxin confers processivity on the DNA polymerase activity of the gene 5 protein of bacteriophage T7. J. Biol. Chem. 262, 16212–16223 (1987)

    CAS  PubMed  Google Scholar 

  27. Hyland, E. M., Rezende, L. F. & Richardson, C. C. The DNA binding domain of the gene 2.5 single-stranded DNA-binding protein of bacteriophage T7. J. Biol. Chem. 278, 7247–7256 (2003)

    Article  CAS  PubMed  Google Scholar 

Download references


We thank J.-B. Lee for technical advice and S. Moskowitz for illustrations. This work was supported by the National Institutes of Health (grants GM-077248 to A.M.v.O. and GM-54397 to C.C.R.) and the National Science Foundation (CAREER grant 0543784 to A.M.v.O.). J.J.L. acknowledges the Jane Coffin Childs Memorial Fund for a postdoctoral fellowship.

Author Contributions S.M.H. performed the single-molecule bead experiments; S.M.H. and J.J.L. performed the single-molecule fluorescence experiments; S.M.H. and M.T. performed the bulk-phase experiments; S.M.H., C.C.R. and A.M.v.O. designed the experiments, analysed the data and wrote the manuscript.

Author information

Authors and Affiliations


Corresponding author

Correspondence to Antoine M. van Oijen.

Supplementary information

Supplementary Information

This file contains Supplementary Methods, Supplementary Figures S1-S8 with Legends and Supplementary References. (PDF 1452 kb)

PowerPoint slides

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Hamdan, S., Loparo, J., Takahashi, M. et al. Dynamics of DNA replication loops reveal temporal control of lagging-strand synthesis. Nature 457, 336–339 (2009).

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI:

This article is cited by


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.


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