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.

Cryo-EM structure of a helicase loading intermediate containing ORC–Cdc6–Cdt1–MCM2-7 bound to DNA

Nature Structural & Molecular Biology volume 20, pages 944–951 (2013)Cite this article

Subjects

Abstract

In eukaryotes, the Cdt1-bound replicative helicase core MCM2-7 is loaded onto DNA by the ORC–Cdc6 ATPase to form a prereplicative complex (pre-RC) with an MCM2-7 double hexamer encircling DNA. Using purified components in the presence of ATP-γS, we have captured in vitro an intermediate in pre-RC assembly that contains a complex between the ORC–Cdc6 and Cdt1–MCM2-7 heteroheptamers called the OCCM. Cryo-EM studies of this 14-subunit complex reveal that the two separate heptameric complexes are engaged extensively, with the ORC–Cdc6 N-terminal AAA+ domains latching onto the C-terminal AAA+ motor domains of the MCM2-7 hexamer. The conformation of ORC–Cdc6 undergoes a concerted change into a right-handed spiral with helical symmetry that is identical to that of the DNA double helix. The resulting ORC–Cdc6 helicase loader shows a notable structural similarity to the replication factor C clamp loader, suggesting a conserved mechanism of action.

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

Prices vary by article type

from$1.95

to$39.95

Learn more

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

Figure 1: In vitro assembly of the OCCM complex.
Figure 2: Cryo-EM of the eukaryotic OCCM complex.
Figure 3: Mapping the protein and DNA components of the OCCM.
Figure 4: Segmented cryo-EM structure of the OCCM.
Figure 5: Cryo-EM structure of the yeast Cdt1–MCM2-7 in the context of the OCCM complex compared with the Drosophila MCM2-7 structure.
Figure 6: Upon recruitment of Cdt1–MCM2-7, ORC–Cdc6 undergoes concerted conformational change into a right-handed spiral structure.
Figure 7: The DNA apparently passes through the middle of the OCCM complex.

Accession codes

Primary accessions

Electron Microscopy Data Bank

Referenced accessions

Electron Microscopy Data Bank

Protein Data Bank

References

  1. Bell, S.P. & Dutta, A. DNA replication in eukaryotic cells. Annu. Rev. Biochem. 71, 333–374 (2002).

    Article  CAS  Google Scholar 

  2. Stillman, B. Origin recognition and the chromosome cycle. FEBS Lett. 579, 877–884 (2005).

    Article  CAS  Google Scholar 

  3. 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 

  4. Remus, D. & Diffley, J.F. Eukaryotic DNA replication control: lock and load, then fire. Curr. Opin. Cell Biol. 21, 771–777 (2009).

    Article  CAS  Google Scholar 

  5. 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 

  6. Clarey, M.G. et al. Nucleotide-dependent conformational changes in the DnaA-like core of the origin recognition complex. Nat. Struct. Mol. Biol. 13, 684–690 (2006).

    Article  CAS  Google Scholar 

  7. Li, H. & Stillman, B. The origin recognition complex: a biochemical and structural view. Subcell. Biochem. 62, 37–58 (2012).

    Article  CAS  Google Scholar 

  8. Santocanale, C. & Diffley, J.F. ORC- and Cdc6-dependent complexes at active and inactive chromosomal replication origins in Saccharomyces cerevisiae. EMBO J. 15, 6671–6679 (1996).

    Article  CAS  Google Scholar 

  9. 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 

  10. 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 

  11. 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 

  12. 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 

  13. Remus, D. et al. Concerted loading of MCM2-7 double hexamers around DNA during DNA replication origin licensing. Cell 139, 719–730 (2009).

    Article  CAS  Google Scholar 

  14. Moyer, S.E., Lewis, P.W. & Botchan, M.R. Isolation of the Cdc45/MCM2-7/GINS (CMG) complex, a candidate for the eukaryotic DNA replication fork helicase. Proc. Natl. Acad. Sci. USA 103, 10236–10241 (2006).

    Article  CAS  Google Scholar 

  15. Ilves, I., Petojevic, T., Pesavento, J.J. & Botchan, M.R. Activation of the MCM2-7 helicase by association with Cdc45 and GINS proteins. Mol. Cell 37, 247–258 (2010).

    Article  CAS  Google Scholar 

  16. Kang, Y.H., Galal, W.C., Farina, A., Tappin, I. & Hurwitz, J. Properties of the human Cdc45/MCM2-7/GINS helicase complex and its action with DNA polymerase epsilon in rolling circle DNA synthesis. Proc. Natl. Acad. Sci. USA 109, 6042–6047 (2012).

    Article  CAS  Google Scholar 

  17. Heller, R.C. et al. Eukaryotic origin-dependent DNA replication in vitro reveals sequential action of DDK and S-CDK kinases. Cell 146, 80–91 (2011).

    Article  CAS  Google Scholar 

  18. 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 

  19. Gaudier, M., Schuwirth, B.S., Westcott, S.L. & Wigley, D.B. Structural basis of DNA replication origin recognition by an ORC protein. Science 317, 1213–1216 (2007).

    Article  CAS  Google Scholar 

  20. Dueber, E.L., Corn, J.E., Bell, S.D. & Berger, J.M. Replication origin recognition and deformation by a heterodimeric archaeal Orc1 complex. Science 317, 1210–1213 (2007).

    Article  CAS  Google Scholar 

  21. Clarey, M.G., Botchan, M. & Nogales, E. Single particle EM studies of the Drosophila melanogaster origin recognition complex and evidence for DNA wrapping. J. Struct. Biol. 164, 241–249 (2008).

    Article  CAS  Google Scholar 

  22. Liu, C. et al. Structural insights into the Cdt1-mediated MCM2-7 chromatin loading. Nucleic Acids Res. 40, 3208–3217 (2012).

    Article  CAS  Google Scholar 

  23. 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 

  24. Onesti, S. & MacNeill, S.A. Structure and evolutionary origins of the CMG complex. Chromosoma 122, 47–53 (2013).

    Article  CAS  Google Scholar 

  25. Forsburg, S.L. Eukaryotic MCM proteins: beyond replication initiation. Microbiol. Mol. Biol. Rev. 68, 109–131 (2004).

    Article  CAS  Google Scholar 

  26. Bochman, M.L. & Schwacha, A. The Mcm complex: unwinding the mechanism of a replicative helicase. Microbiol. Mol. Biol. Rev. 73, 652–683 (2009).

    Article  CAS  Google Scholar 

  27. Brewster, A.S. et al. Crystal structure of a near-full-length archaeal MCM: functional insights for an AAA+ hexameric helicase. Proc. Natl. Acad. Sci. USA 105, 20191–20196 (2008).

    Article  CAS  Google Scholar 

  28. Chesnokov, I.N., Chesnokova, O.N. & Botchan, M. A cytokinetic function of Drosophila ORC6 protein resides in a domain distinct from its replication activity. Proc. Natl. Acad. Sci. USA 100, 9150–9155 (2003).

    Article  CAS  Google Scholar 

  29. Khayrutdinov, B.I. et al. Structure of the Cdt1 C-terminal domain: conservation of the winged helix fold in replication licensing factors. Protein Sci. 18, 2252–2264 (2009).

    Article  CAS  Google Scholar 

  30. 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 

  31. Pape, T. et al. Hexameric ring structure of the full-length archaeal MCM protein complex. EMBO Rep. 4, 1079–1083 (2003).

    Article  CAS  Google Scholar 

  32. Suck, D. & Oefner, C. Structure of DNase I at 2.0-Å resolution suggests a mechanism for binding to and cutting DNA. Nature 321, 620–625 (1986).

    Article  CAS  Google Scholar 

  33. Henderson, R. et al. Tilt-pair analysis of images from a range of different specimens in single-particle electron cryomicroscopy. J. Mol. Biol. 413, 1028–1046 (2011).

    Article  CAS  Google Scholar 

  34. Chen, Z. et al. The architecture of the DNA replication origin recognition complex in Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA 105, 10326–10331 (2008).

    Article  CAS  Google Scholar 

  35. Li, H., Chavan, M., Schindelin, H., Lennarz, W.J. & Li, H. Structure of the oligosaccharyl transferase complex at 12-Å resolution. Structure 16, 432–440 (2008).

    Article  CAS  Google Scholar 

  36. Lander, G.C. et al. Complete subunit architecture of the proteasome regulatory particle. Nature 482, 186–191 (2012).

    Article  CAS  Google Scholar 

  37. Lau, P.W., Potter, C.S., Carragher, B. & MacRae, I.J. DOLORS: versatile strategy for internal labeling and domain localization in electron microscopy. Structure 20, 1995–2002 (2012).

    Article  CAS  Google Scholar 

  38. Bochman, M.L., Bell, S.P. & Schwacha, A. Subunit organization of MCM2-7 and the unequal role of active sites in ATP hydrolysis and viability. Mol. Cell Biol. 28, 5865–5873 (2008).

    Article  CAS  Google Scholar 

  39. Pintilie, G.D., Zhang, J., Goddard, T.D., Chiu, W. & Gossard, D.C. Quantitative analysis of cryo-EM density map segmentation by watershed and scale-space filtering, and fitting of structures by alignment to regions. J. Struct. Biol. 170, 427–438 (2010).

    Article  CAS  Google Scholar 

  40. 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 

  41. Liu, J. et al. Structure and function of Cdc6/Cdc18: implications for origin recognition and checkpoint control. Mol. Cell 6, 637–648 (2000).

    Article  CAS  Google Scholar 

  42. 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 

  43. Frigola, J., Remus, D., Mehanna, A. & Diffley, J.F. ATPase-dependent quality control of DNA replication origin licensing. Nature 495, 339–343 (2013).

    Article  CAS  Google Scholar 

  44. 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).

    Article  Google Scholar 

  45. 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, 3162–3167 (2013).

    Article  CAS  Google Scholar 

  46. Kawasaki, Y., Kim, H.D., Kojima, A., Seki, T. & Sugino, A. Reconstitution of Saccharomyces cerevisiae prereplicative complex assembly in vitro. Genes Cells 11, 745–756 (2006).

    Article  CAS  Google Scholar 

  47. Ishimi, Y. A DNA helicase activity is associated with an MCM4, -6, and -7 protein complex. J. Biol. Chem. 272, 24508–24513 (1997).

    Article  CAS  Google Scholar 

  48. Bochman, M.L. & Schwacha, A. Differences in the single-stranded DNA binding activities of MCM2-7 and MCM467: MCM2 and MCM5 define a slow ATP-dependent step. J. Biol. Chem. 282, 33795–33804 (2007).

    Article  CAS  Google Scholar 

  49. Waga, S. & Stillman, B. The DNA replication fork in eukaryotic cells. Annu. Rev. Biochem. 67, 721–751 (1998).

    Article  CAS  Google Scholar 

  50. Kelch, B.A., Makino, D.L., O'Donnell, M. & Kuriyan, J. How a DNA polymerase clamp loader opens a sliding clamp. Science 334, 1675–1680 (2011).

    Article  CAS  Google Scholar 

  51. Bowman, G.D., O'Donnell, M. & Kuriyan, J. Structural analysis of a eukaryotic sliding DNA clamp-clamp loader complex. Nature 429, 724–730 (2004).

    Article  CAS  Google Scholar 

  52. O'Donnell, M. & Kuriyan, J. Clamp loaders and replication initiation. Curr. Opin. Struct. Biol. 16, 35–41 (2006).

    Article  CAS  Google Scholar 

  53. Speck, C. & Stillman, B. Cdc6 ATPase activity regulates ORC–Cdc6 stability and the selection of specific DNA sequences as origins of DNA replication. J. Biol. Chem. 282, 11705–11714 (2007).

    Article  CAS  Google Scholar 

  54. Miyata, T. et al. Open clamp structure in the clamp-loading complex visualized by electron microscopic image analysis. Proc. Natl. Acad. Sci. USA 102, 13795–13800 (2005).

    Article  CAS  Google Scholar 

  55. 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).

    Article  CAS  Google Scholar 

  56. Zou, L. & Stillman, B. Assembly of a complex containing Cdc45p, replication protein A, and Mcm2p at replication origins controlled by S-phase cyclin-dependent kinases and Cdc7p-Dbf4p kinase. Mol. Cell Biol. 20, 3086–3096 (2000).

    Article  CAS  Google Scholar 

  57. 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 

  58. Baker, M.L., Zhang, J., Ludtke, S.J. & Chiu, W. Cryo-EM of macromolecular assemblies at near-atomic resolution. Nat. Protoc. 5, 1697–1708 (2010).

    Article  CAS  Google Scholar 

  59. Tang, G. et al. EMAN2: an extensible image processing suite for electron microscopy. J. Struct. Biol. 157, 38–46 (2007).

    Article  CAS  Google Scholar 

  60. Pettersen, E.F. et al. UCSF Chimera—a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank M. Smulczeski and S. Zhang for helping to manually select a large number of particles from raw cryo-EM micrographs and E. Gardenal and C. Winkler for the MCM2-7–Cdc6 interaction analysis. This work was supported by US National Institutes of Health grants GM45436 (to B.S.) and GM74985 (to H.L.) and the United Kingdom Medical Research Council (to C.S.). H.K. was supported by Postdoctoral Fellowships for Research Abroad from the Japan Society for the Promotion of Science and the Uehara Memorial Foundation.

Author information

Author notes

  1. Hironori Kawakami

    Present address: Present address: Department of Molecular Biology, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka, Japan.,

  2. Jingchuan Sun and Cecile Evrin: These authors contributed equally to this work.

Authors and Affiliations

  1. Biosciences Department, Brookhaven National Laboratory, Upton, New York, USA

    Jingchuan Sun & Huilin Li

  2. DNA Replication Group, MRC Clinical Sciences Centre, Imperial College Faculty of Medicine, Hammersmith Hospital Campus, London, UK

    Cecile Evrin, Stefan A Samel, Alejandra Fernández-Cid, Alberto Riera & Christian Speck

  3. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, USA

    Hironori Kawakami & Bruce Stillman

  4. Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, New York, USA

    Huilin Li

Authors
  1. Jingchuan Sun
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Cecile Evrin
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Stefan A Samel
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Alejandra Fernández-Cid
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Alberto Riera
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Hironori Kawakami
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Bruce Stillman
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Christian Speck
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Huilin Li
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

J.S., C.E., S.A.S., A.F.-C., A.R. and H.K. performed the specimen preparation and biochemistry. J.S. collected the cryo-EM data, performed the cryo-EM reconstructions. J.S., B.S., C.S. and H.L. designed experiments and wrote the manuscript.

Corresponding authors

Correspondence to Bruce Stillman, Christian Speck or Huilin Li.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–5 (PDF 675 kb)

Supplementary Video 1

Surface-rendered cryo-EM 3D map of the OCCM complex. Display threshold is set to include the expected mass of ~ 1.1 MDa. (MP4 2077 kb)

Supplementary Video 2

Segmented 3D density of the OCCM complex. Each of the 14 protein subunits of the complex is shown in a different color. The gray density may contain the dsDNA. (MP4 2343 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Sun, J., Evrin, C., Samel, S. 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). https://doi.org/10.1038/nsmb.2629

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nsmb.2629

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