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.

Coordinating DNA replication by means of priming loop and differential synthesis rate


Genomic DNA is replicated by two DNA polymerase molecules, one of which works in close association with the helicase to copy the leading-strand template in a continuous manner while the second copies the already unwound lagging-strand template in a discontinuous manner through the synthesis of Okazaki fragments1,2. Considering that the lagging-strand polymerase has to recycle after the completion of every Okazaki fragment through the slow steps of primer synthesis and hand-off to the polymerase3,4,5, it is not understood how the two strands are synthesized with the same net rate6,7,8,9. Here we show, using the T7 replication proteins10,11, that RNA primers are made ‘on the fly’ during ongoing DNA synthesis and that the leading-strand T7 replisome does not pause during primer synthesis, contrary to previous reports12,13. Instead, the leading-strand polymerase remains limited by the speed of the helicase14; it therefore synthesizes DNA more slowly than the lagging-strand polymerase. We show that the primase–helicase T7 gp4 maintains contact with the priming sequence during ongoing DNA synthesis; the nascent lagging-strand template therefore organizes into a priming loop that keeps the primer in physical proximity to the replication complex. Our findings provide three synergistic mechanisms of coordination: first, primers are made concomitantly with DNA synthesis; second, the priming loop ensures efficient primer use and hand-off to the polymerase; and third, the lagging-strand polymerase copies DNA faster, which allows it to keep up with leading-strand DNA synthesis overall.

Your institute does not have access to this article

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Figure 1: Primer synthesis occurs concomitantly with DNA unwinding and synthesis.
Figure 2: Priming loop: the primase domain maintains contact with the priming sequence during replication.
Figure 3: Lagging-strand synthesis is faster than leading-strand synthesis.
Figure 4: Model of T7 DNA replication.


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

    CAS  Article  Google Scholar 

  2. O’Donnell, M. Replisome architecture and dynamics in Escherichia coli . J. Biol. Chem. 281, 10653–10656 (2006)

    Article  Google Scholar 

  3. Stukenberg, P. T., Turner, J. & O’Donnell, M. An explanation for lagging strand replication: polymerase hopping among DNA sliding clamps. Cell 78, 877–887 (1994)

    CAS  Article  Google Scholar 

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

    CAS  Article  Google Scholar 

  5. Patel, S. S., Hingorani, M. M. & Ng, W. M. The K318A mutant of bacteriophage T7 DNA primase–helicase protein is deficient in helicase but not primase activity and inhibits primase–helicase protein wild-type activities by heterooligomer formation. Biochemistry 33, 7857–7868 (1994)

    CAS  Article  Google Scholar 

  6. 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  Google Scholar 

  7. Wu, C. A., Zechner, E. L. & Marians, K. J. Coordinated leading- and lagging-strand synthesis at the Escherichia coli DNA replication fork. I. Multiple effectors act to modulate Okazaki fragment size. J. Biol. Chem. 267, 4030–4044 (1992)

    CAS  PubMed  Google Scholar 

  8. 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)

    CAS  Article  Google Scholar 

  9. Salinas, F. & Benkovic, S. J. Characterization of bacteriophage T4-coordinated leading- and lagging-strand synthesis on a minicircle substrate. Proc. Natl Acad. Sci. USA 97, 7196–7201 (2000)

    ADS  CAS  Article  Google Scholar 

  10. Donmez, I. & Patel, S. S. Mechanisms of a ring shaped helicase. Nucleic Acids Res. 34, 4216–4224 (2006)

    CAS  Article  Google Scholar 

  11. Hamdan, S. M. & Richardson, C. C. Motors, switches, and contacts in the replisome. Annu. Rev. Biochem. 78, 205–243 (2009)

    CAS  Article  Google Scholar 

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

    ADS  CAS  Article  Google Scholar 

  13. 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)

    ADS  CAS  Article  Google Scholar 

  14. Stano, N. M. et al. DNA synthesis provides the driving force to accelerate DNA unwinding by a helicase. Nature 435, 370–373 (2005)

    ADS  CAS  Article  Google Scholar 

  15. Donmez, I. & Patel, S. S. Coupling of DNA unwinding to nucleotide hydrolysis in a ring-shaped helicase. EMBO J. 27, 1718–1726 (2008)

    CAS  Article  Google Scholar 

  16. Jeong, Y. J., Levin, M. K. & Patel, S. S. The DNA-unwinding mechanism of the ring helicase of bacteriophage T7. Proc. Natl Acad. Sci. USA 101, 7264–7269 (2004)

    ADS  CAS  Article  Google Scholar 

  17. Ha, T. et al. Initiation and re-initiation of DNA unwinding by the Escherichia coli Rep helicase. Nature 419, 638–641 (2002)

    ADS  CAS  Article  Google Scholar 

  18. Yang, J., Trakselis, M. A., Roccasecca, R. M. & Benkovic, S. J. The application of a minicircle substrate in the study of the coordinated T4 DNA replication. J. Biol. Chem. 278, 49828–49838 (2003)

    CAS  Article  Google Scholar 

  19. 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)

    ADS  CAS  Article  Google Scholar 

  20. Corn, J. E., Pelton, J. G. & Berger, J. M. Identification of a DNA primase template tracking site redefines the geometry of primer synthesis. Nature Struct. Mol. Biol. 15, 163–169 (2008)

    CAS  Article  Google Scholar 

  21. Myong, S. et al. Cytosolic viral sensor RIG-I is a 5′-triphosphate-dependent translocase on double-stranded RNA. Science 323, 1070–1074 (2009)

    ADS  CAS  Article  Google Scholar 

  22. Sanborn, M. E., Connolly, B. K., Gurunathan, K. & Levitus, M. Fluorescence properties and photophysics of the sulfoindocyanine Cy3 linked covalently to DNA. J. Phys. Chem. B 111, 11064–11074 (2007)

    CAS  Article  Google Scholar 

  23. Liu, S., Abbondanzieri, E. A., Rausch, J. W., Le Grice, S. F. & Zhuang, X. Slide into action: dynamic shuttling of HIV reverse transcriptase on nucleic acid substrates. Science 322, 1092–1097 (2008)

    ADS  CAS  Article  Google Scholar 

  24. Selick, H. E. et al. in DNA Replication and Recombination (eds McMacken, R. & Kelly, T. J.) 183–214 (Alan R. Liss Inc., 1987)

    Google Scholar 

  25. Hamdan, S. M. et al. A unique loop in T7 DNA polymerase mediates the binding of helicase-primase, DNA binding protein, and processivity factor. Proc. Natl Acad. Sci. USA 102, 5096–5101 (2005)

    ADS  CAS  Article  Google Scholar 

  26. McInerney, P., Johnson, A., Katz, F. & O’Donnell, M. Characterization of a triple DNA polymerase replisome. Mol. Cell 27, 527–538 (2007)

    CAS  Article  Google Scholar 

  27. 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)

    CAS  Article  Google Scholar 

  28. Patel, S. S., Rosenberg, A. H., Studier, F. W. & Johnson, K. A. Large scale purification and biochemical characterization of T7 primase/helicase proteins. Evidence for homodimer and heterodimer formation. J. Biol. Chem. 267, 15013–15021 (1992)

    CAS  PubMed  Google Scholar 

  29. Patel, S. S., Wong, I. & Johnson, K. A. Pre-steady-state kinetic analysis of processive DNA replication including complete characterization of an exonuclease-deficient mutant. Biochemistry 30, 511–525 (1991)

    CAS  Article  Google Scholar 

  30. Ha, T. Single-molecule fluorescence resonance energy transfer. Methods 25, 78–86 (2001)

    CAS  Article  Google Scholar 

  31. Rasnik, I., McKinney, S. A. & Ha, T. Nonblinking and long-lasting single-molecule fluorescence imaging. Nature Methods 3, 891–893 (2006)

    CAS  Article  Google Scholar 

Download references


We thank C. M. Drain for a critical reading of the paper, K. Picha and S. Patel for preparation of the minicircle DNA, and S. Arslan and K. S. Lee for help with single-molecule FRET data analysis. This work was supported by NIH grants GM55310 (S.S.P.) and GM065367 (T.H.) and NSF grants 0822613 and 0646550 (T.H.). T.H. is an investigator with the Howard Hughes Medical Institute.

Author Contributions M.P. purified T7 gp5 and T7 gp4, and constructed DNA substrates for the priming-loop studies, and obtained and analysed all the ensemble DNA synthesis and primer synthesis experiments. S.S. developed robust single-molecule assays for observing DNA unwinding and priming-loop formation, and obtained and analysed all single-molecule data. I.D. and G.P. performed the ensemble unwinding experiments. M.P., S.S., S.S.P. and T.H. designed the experiments, analysed the data and wrote the manuscript.

Author information

Authors and Affiliations


Corresponding authors

Correspondence to Taekjip Ha or Smita S. Patel.

Supplementary information

Supplementary Information

This file contains Supplementary Methods, Supplementary Tables 1-3, Supplementary Figures 1-13 with Legends and Supplementary References. (PDF 2040 kb)

PowerPoint slides

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Pandey, M., Syed, S., Donmez, I. et al. Coordinating DNA replication by means of priming loop and differential synthesis rate . Nature 462, 940–943 (2009).

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI:

Further reading


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