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.

  • Perspective
  • Published:

Prereplication-complex formation: a molecular double take?

Nature Structural & Molecular Biology volume 21, pages 20–25 (2014)Cite this article

Subjects

Abstract

In G1, two copies of the MCM2–7 helicase are recruited to each origin of replication. Whereas recruitment of the first MCM2–7 is likely to be analogous to the loading of sliding clamps around DNA, how the second MCM2–7 complex is recruited is highly contentious. Here, we argue that MCM2–7 loading involves specific modifications to the clamp-loading reaction and propose that the first and second MCM2–7 molecules are loaded via similar mechanisms.

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

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

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

Figure 1: The mechanism of helicase loading in bacteria.
Figure 2: The mechanism of clamp loading.
Figure 3: Binding of the first MCM2–7 to the origin.
Figure 4: Possible mechanisms for binding of the second MCM2–7 to the origin.

Similar content being viewed by others

References

  1. Bell, S.P. & Kaguni, J.M. Helicase loading at chromosomal origins of replication. Cold Spring Harb. Perspect. Biol. 5, a010124 (2013).

    PubMed  PubMed Central  Google Scholar 

  2. Mott, M.L., Erzberger, J.P., Coons, M.M. & Berger, J.M. Structural synergy and molecular crosstalk between bacterial helicase loaders and replication initiators. Cell 135, 623–634 (2008).

    Article  CAS  Google Scholar 

  3. Siddiqui, K., On, K.F. & Diffley, J.F. Regulating DNA replication in eukarya. Cold Spring Harb. Perspect. Biol. 5, a012930 (2013).

    Article  Google Scholar 

  4. Boos, D., Frigola, J. & Diffley, J.F. Activation of the replicative DNA helicase: breaking up is hard to do. Curr. Opin. Cell Biol. 24, 423–430 (2012).

    Article  CAS  Google Scholar 

  5. Fu, Y.V. et al. Selective bypass of a lagging strand roadblock by the eukaryotic replicative DNA helicase. Cell 146, 931–941 (2011).

    Article  CAS  Google Scholar 

  6. Tanaka, S. & Araki, H. Helicase activation and establishment of replication forks at chromosomal origins of replication. Cold Spring Harb. Perspect. Biol. doi:10.1101/cshperspect.a010371 (23 July 2013).

  7. Arias, E.E. & Walter, J.C. Strength in numbers: preventing rereplication via multiple mechanisms in eukaryotic cells. Genes Dev. 21, 497–518 (2007).

    Article  CAS  Google Scholar 

  8. Kelch, B.A., Makino, D.L., O'Donnell, M. & Kuriyan, J. Clamp loader ATPases and the evolution of DNA replication machinery. BMC Biol. 10, 34 (2012).

    Article  CAS  Google Scholar 

  9. Arias-Palomo, E., O'Shea, V.L., Hood, I.V. & Berger, J.M. The bacterial DnaC helicase loader is a DnaB ring breaker. Cell 153, 438–448 (2013). This structure of the DnaB–DnaC complex shows how DnaC cracks open the DnaB ring and illustrates the similarity between the loading mechanism of the bacterial helicase and the sliding clamp.

    Article  CAS  Google Scholar 

  10. Bailey, S., Eliason, W.K. & Steitz, T.A. Structure of hexameric DnaB helicase and its complex with a domain of DnaG primase. Science 318, 459–463 (2007).

    Article  CAS  Google Scholar 

  11. Galletto, R., Maillard, R., Jezewska, M.J. & Bujalowski, W. Global conformation of the Escherichia coli replication factor DnaC protein in absence and presence of nucleotide cofactors. Biochemistry 43, 10988–11001 (2004).

    Article  CAS  Google Scholar 

  12. Davey, M.J., Fang, L., McInerney, P., Georgescu, R.E. & O'Donnell, M. The DnaC helicase loader is a dual ATP/ADP switch protein. EMBO J. 21, 3148–3159 (2002).

    Article  CAS  Google Scholar 

  13. Ludlam, A.V., McNatt, M.W., Carr, K.M. & Kaguni, J.M. Essential amino acids of Escherichia coli DnaC protein in an N-terminal domain interact with DnaB helicase. J. Biol. Chem. 276, 27345–27353 (2001).

    Article  CAS  Google Scholar 

  14. Bell, S.P. & Stillman, B. ATP-dependent recognition of eukaryotic origins of DNA replication by a multiprotein complex. Nature 357, 128–134 (1992).

    Article  CAS  Google Scholar 

  15. Remus, D., Beall, E.L. & Botchan, M.R. DNA topology, not DNA sequence, is a critical determinant for Drosophila ORC-DNA binding. EMBO J. 23, 897–907 (2004).

    Article  CAS  Google Scholar 

  16. Vashee, S. et al. Sequence-independent DNA binding and replication initiation by the human origin recognition complex. Genes Dev. 17, 1894–1908 (2003).

    Article  CAS  Google Scholar 

  17. Leonard, A.C. & Mechali, M. DNA replication origins. Cold Spring Harb. Perspect. Biol. 5, a010116 (2013).

    Article  Google Scholar 

  18. Bell, S.P., Mitchell, J., Leber, J., Kobayashi, R. & Stillman, B. The multidomain structure of Orc1p reveals similarity to regulators of DNA replication and transcriptional silencing. Cell 83, 563–568 (1995).

    Article  CAS  Google Scholar 

  19. Perkins, G. & Diffley, J.F. Nucleotide-dependent prereplicative complex assembly by Cdc6p, a homolog of eukaryotic and prokaryotic clamp-loaders. Mol. Cell 2, 23–32 (1998).

    Article  CAS  Google Scholar 

  20. Speck, C., Chen, Z., Li, H. & Stillman, B. ATPase-dependent cooperative binding of ORC and Cdc6 to origin DNA. Nat. Struct. Mol. Biol. 12, 965–971 (2005).

    Article  CAS  Google Scholar 

  21. Klemm, R.D., Austin, R.J. & Bell, S.P. Coordinate binding of ATP and origin DNA regulates the ATPase activity of the origin recognition complex. Cell 88, 493–502 (1997).

    Article  CAS  Google Scholar 

  22. Bowers, J.L., Randell, J.C., Chen, S. & Bell, S.P. ATP hydrolysis by ORC catalyzes reiterative Mcm2–7 assembly at a defined origin of replication. Mol. Cell 16, 967–978 (2004).

    Article  CAS  Google Scholar 

  23. Liu, S. et al. Structural analysis of human Orc6 protein reveals a homology with transcription factor TFIIB. Proc. Natl. Acad. Sci. USA 108, 7373–7378 (2011).

    Article  CAS  Google Scholar 

  24. Bleichert, F. et al. A Meier-Gorlin syndrome mutation in a conserved C-terminal helix of Orc6 impedes origin recognition complex formation. eLife 2, e00882 (2013).

    Article  Google Scholar 

  25. Klemm, R.D. & Bell, S.P. ATP bound to the origin recognition complex is important for preRC formation. Proc. Natl. Acad. Sci. USA 98, 8361–8367 (2001).

    Article  CAS  Google Scholar 

  26. Sun, J. et al. Cdc6-induced conformational changes in ORC bound to origin DNA revealed by cryo-electron microscopy. Structure 20, 534–544 (2012).

    Article  CAS  Google Scholar 

  27. Costa, A. et al. The structural basis for MCM2–7 helicase activation by GINS and Cdc45. Nat. Struct. Mol. Biol. 18, 471–477 (2011).

    Article  CAS  Google Scholar 

  28. Fletcher, R.J. et al. The structure and function of MCM from archaeal M. thermoautotrophicum. Nat. Struct. Biol. 10, 160–167 (2003).

    Article  CAS  Google Scholar 

  29. McGeoch, A.T., Trakselis, M.A., Laskey, R.A. & Bell, S.D. Organization of the archaeal MCM complex on DNA and implications for the helicase mechanism. Nat. Struct. Mol. Biol. 12, 756–762 (2005).

    Article  CAS  Google Scholar 

  30. Remus, D. et al. Concerted loading of Mcm2–7 double hexamers around DNA during DNA replication origin licensing. Cell 139, 719–730 (2009). Together with Evrin et al.38, this paper shows that MCM2–7 complexes are loaded as highly stable double hexamers during pre-RC formation.

    Article  CAS  Google Scholar 

  31. Bochman, M.L. & Schwacha, A. The Mcm2–7 complex has in vitro helicase activity. Mol. Cell 31, 287–293 (2008).

    Article  CAS  Google Scholar 

  32. Tanaka, S. & Diffley, J.F. Interdependent nuclear accumulation of budding yeast Cdt1 and Mcm2–7 during G1 phase. Nat. Cell Biol. 4, 198–207 (2002).

    Article  CAS  Google Scholar 

  33. Fernández-Cid, A. et al. An ORC/Cdc6/MCM2–7 complex is formed in a multistep reaction to serve as a platform for MCM double-hexamer assembly. Mol. Cell 50, 577–588 (2013). This paper shows that pre-RC formation involves two temporally separable rounds of MCM2–7 loading.

    Article  Google Scholar 

  34. Zhang, J. et al. The interacting domains of hCdt1 and hMcm6 involved in the chromatin loading of the MCM complex in human cells. Cell Cycle 9, 4848–4857 (2010).

    Article  CAS  Google Scholar 

  35. Sun, J. et al. Cryo-EM structure of a helicase loading intermediate containing ORC–Cdc6–Cdt1–MCM2-7 bound to DNA. Nat. Struct. Mol. Biol. 20, 944–951 (2013). This paper suggests that ORC–Cdc6 binds to MCM2–7–Cdt1 analogously to the binding of clamp loaders to clamps.

    Article  CAS  Google Scholar 

  36. Frigola, J., Remus, D., Mehanna, A. & Diffley, J.F. ATPase-dependent quality control of DNA replication origin licensing. Nature 495, 339–343 (2013). This paper illustrates how ATP hydrolysis aborts nonproductive pre-RC assembly reactions.

    Article  CAS  Google Scholar 

  37. Donovan, S., Harwood, J., Drury, L.S. & Diffley, J.F. Cdc6p-dependent loading of Mcm proteins onto pre-replicative chromatin in budding yeast. Proc. Natl. Acad. Sci. USA 94, 5611–5616 (1997).

    Article  CAS  Google Scholar 

  38. Evrin, C. et al. A double-hexameric MCM2–7 complex is loaded onto origin DNA during licensing of eukaryotic DNA replication. Proc. Natl. Acad. Sci. USA 106, 20240–20245 (2009).

    Article  CAS  Google Scholar 

  39. Gambus, A., Khoudoli, G.A., Jones, R.C. & Blow, J.J. MCM2–7 form double hexamers at licensed origins in Xenopus egg extract. J. Biol. Chem. 286, 11855–11864 (2011).

    Article  CAS  Google Scholar 

  40. Randell, J.C., Bowers, J.L., Rodriguez, H.K. & Bell, S.P. Sequential ATP hydrolysis by Cdc6 and ORC directs loading of the Mcm2–7 helicase. Mol. Cell 21, 29–39 (2006).

    Article  CAS  Google Scholar 

  41. Evrin, C. et al. In the absence of ATPase activity, pre-RC formation is blocked prior to MCM2–7 hexamer dimerization. Nucleic Acids Res. 41, 3162–3172 (2013).

    Article  CAS  Google Scholar 

  42. Gillespie, P.J., Li, A. & Blow, J.J. Reconstitution of licensed replication origins on Xenopus sperm nuclei using purified proteins. BMC Biochem. 2, 15 (2001).

    Article  CAS  Google Scholar 

  43. Ying, C.Y. & Gautier, J. The ATPase activity of MCM2–7 is dispensable for pre-RC assembly but is required for DNA unwinding. EMBO J. 24, 4334–4344 (2005).

    Article  CAS  Google Scholar 

  44. Chen, S. & Bell, S.P. CDK prevents Mcm2–7 helicase loading by inhibiting Cdt1 interaction with Orc6. Genes Dev. 25, 363–372 (2011).

    Article  CAS  Google Scholar 

  45. Lyubimov, A.Y., Costa, A., Bleichert, F., Botchan, M.R. & Berger, J.M. ATP-dependent conformational dynamics underlie the functional asymmetry of the replicative helicase from a minimalist eukaryote. Proc. Natl. Acad. Sci. USA 109, 11999–12004 (2012).

    Article  CAS  Google Scholar 

  46. Takara, T.J. & Bell, S.P. Multiple Cdt1 molecules act at each origin to load replication-competent Mcm2–7 helicases. EMBO J. 30, 4885–4896 (2011).

    Article  CAS  Google Scholar 

  47. Samson, R.Y. & Bell, S.D. MCM loading: an open-and-shut case? Mol. Cell 50, 457–458 (2013).

    Article  CAS  Google Scholar 

  48. Chen, S., de Vries, M.A. & Bell, S.P. Orc6 is required for dynamic recruitment of Cdt1 during repeated Mcm2–7 loading. Genes Dev. 21, 2897–2907 (2007).

    Article  CAS  Google Scholar 

  49. Takahashi, T., Ohara, E., Nishitani, H. & Masukata, H. Multiple ORC-binding sites are required for efficient MCM loading and origin firing in fission yeast. EMBO J. 22, 964–974 (2003).

    Article  CAS  Google Scholar 

  50. Austin, R.J., Orr-Weaver, T.L. & Bell, S.P. Drosophila ORC specifically binds to ACE3, an origin of DNA replication control element. Genes Dev. 13, 2639–2649 (1999).

    Article  CAS  Google Scholar 

  51. Edgar, B.A. & Orr-Weaver, T.L. Endoreplication cell cycles: more for less. Cell 105, 297–306 (2001).

    Article  CAS  Google Scholar 

  52. Eaton, M.L., Galani, K., Kang, S., Bell, S.P. & MacAlpine, D.M. Conserved nucleosome positioning defines replication origins. Genes Dev. 24, 748–753 (2010).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

Work on DNA replication in the Walter laboratory is funded by US National Institutes of Health grants GM62267 and GM80676. We thank the many colleagues with whom we discussed mechanisms of pre-RC assembly.

Author information

Author notes

  1. Hasan Yardimci

    Present address: Present address: Cancer Research UK, London Research Institute, Clare Hall Laboratories, South Mimms, UK.,

Authors and Affiliations

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

    Hasan Yardimci & Johannes C Walter

Authors
  1. Hasan Yardimci
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Johannes C Walter
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Johannes C Walter.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Yardimci, H., Walter, J. Prereplication-complex formation: a molecular double take?. Nat Struct Mol Biol 21, 20–25 (2014). https://doi.org/10.1038/nsmb.2738

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nsmb.2738

This article is cited by

Search

Advanced 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