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.

  • Article
  • Published:

The structural basis for MCM2–7 helicase activation by GINS and Cdc45

Nature Structural & Molecular Biology volume 18, pages 471–477 (2011)Cite this article

Subjects

Abstract

Two central steps for initiating eukaryotic DNA replication involve loading of the Mcm2–7 helicase onto double-stranded DNA and its activation by GINS–Cdc45. To better understand these events, we determined the structures of Mcm2–7 and the CMG complex by using single-particle electron microscopy. Mcm2–7 adopts two conformations—a lock-washer-shaped spiral state and a planar, gapped-ring form—in which Mcm2 and Mcm5 flank a breach in the helicase perimeter. GINS and Cdc45 bridge this gap, forming a topologically closed assembly with a large interior channel; nucleotide binding further seals off the discontinuity between Mcm2 and Mcm5, partitioning the channel into two smaller pores. Together, our data help explain how GINS and Cdc45 activate Mcm2–7, indicate that Mcm2–7 loading may be assisted by a natural predisposition of the hexamer to form open rings, and suggest a mechanism by which the CMG complex assists DNA strand separation.

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: Mcm2–7 exists in two states.
Figure 2: Subunit mapping of Mcm2–7.
Figure 3: CMG contains a notched, planar Mcm2–7 ring that is sealed upon nucleotide binding.
Figure 4: Mcm-subunit mapping in the CMG.
Figure 5: Structure docking into CMG reconstructions.
Figure 6: GINS contacts within the CMG.
Figure 7: CMG interactions.
Figure 8: Model for Mcm2–7 activation and function.

Similar content being viewed by others

References

  1. Davey, M.J. & O'Donnell, M. Replicative helicase loaders: ring breakers and ring makers. Curr. Biol. 13, R594–R596 (2003).

    Article  CAS  PubMed  Google Scholar 

  2. Funnell, B.E., Baker, T.A. & Kornberg, A. In vitro assembly of a prepriming complex at the origin of the Escherichia coli chromosome. J. Biol. Chem. 262, 10327–10334 (1987).

    CAS  PubMed  Google Scholar 

  3. 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  PubMed  PubMed Central  Google Scholar 

  4. Valle, M., Gruss, C., Halmer, L., Carazo, J.M. & Donate, L.E. Large T-antigen double hexamers imaged at the simian virus 40 origin of replication. Mol. Cell. Biol. 20, 34–41 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Kumar, A. et al. Model for T-antigen-dependent melting of the simian virus 40 core origin based on studies of the interaction of the beta-hairpin with DNA. J. Virol. 81, 4808–4818 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Schuck, S. & Stenlund, A. Assembly of a double hexameric helicase. Mol. Cell 20, 377–389 (2005).

    Article  CAS  PubMed  Google Scholar 

  7. Enemark, E.J. & Joshua-Tor, L. Mechanism of DNA translocation in a replicative hexameric helicase. Nature 442, 270–275 (2006).

    Article  CAS  PubMed  Google Scholar 

  8. 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  PubMed  Google Scholar 

  9. 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  PubMed  PubMed Central  Google Scholar 

  10. 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  PubMed  PubMed Central  Google Scholar 

  11. 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  PubMed  PubMed Central  Google Scholar 

  12. Gambus, A. et al. GINS maintains association of Cdc45 with MCM in replisome progression complexes at eukaryotic DNA replication forks. Nat. Cell Biol. 8, 358–366 (2006).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  14. Calzada, A., Hodgson, B., Kanemaki, M., Bueno, A. & Labib, K. Molecular anatomy and regulation of a stable replisome at a paused eukaryotic DNA replication fork. Genes Dev. 19, 1905–1919 (2005).

    Article  CAS  PubMed  PubMed Central  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  PubMed  PubMed Central  Google Scholar 

  16. Pacek, M., Tutter, A.V., Kubota, Y., Takisawa, H. & Walter, J.C. Localization of MCM2–7, Cdc45, and GINS to the site of DNA unwinding during eukaryotic DNA replication. Mol. Cell 21, 581–587 (2006).

    Article  CAS  PubMed  Google Scholar 

  17. MacNeill, S.A. Structure and function of the GINS complex, a key component of the eukaryotic replisome. Biochem. J. 425, 489–500 (2010).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  19. 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  PubMed  Google Scholar 

  20. Iyer, L.M., Leipe, D.D., Koonin, E.V. & Aravind, L. Evolutionary history and higher order classification of AAA+ ATPases. J. Struct. Biol. 146, 11–31 (2004).

    Article  CAS  PubMed  Google Scholar 

  21. Davey, M.J., Indiani, C. & O'Donnell, M. Reconstitution of the Mcm2–7p heterohexamer, subunit arrangement, and ATP site architecture. J. Biol. Chem. 278, 4491–4499 (2003).

    Article  CAS  PubMed  Google Scholar 

  22. Crevel, G., Ivetic, A., Ohno, K., Yamaguchi, M. & Cotterill, S. Nearest neighbour analysis of MCM protein complexes in Drosophila melanogaster. Nucleic Acids Res. 29, 4834–4842 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. 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  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. 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  PubMed  PubMed Central  Google Scholar 

  26. Bae, B. et al. Insights into the architecture of the replicative helicase from the structure of an archaeal MCM homolog. Structure 17, 211–222 (2009).

    Article  CAS  PubMed  Google Scholar 

  27. Yu, X. et al. The Methanobacterium thermoautotrophicum MCM protein can form heptameric rings. EMBO Rep. 3, 792–797 (2002).

    Article  PubMed  PubMed Central  Google Scholar 

  28. Gómez-Llorente, Y., Fletcher, R.J., Chen, X.S., Carazo, J.M. & San Martin, C. Polymorphism and double hexamer structure in the archaeal minichromosome maintenance (MCM) helicase from Methanobacterium thermoautotrophicum. J. Biol. Chem. 280, 40909–40915 (2005).

    Article  PubMed  Google Scholar 

  29. Costa, A. et al. Structural basis of the Methanothermobacter thermautotrophicus MCM helicase activity. Nucleic Acids Res. 34, 5829–5838 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Chong, J.P., Hayashi, M.K., Simon, M.N., Xu, R.M. & Stillman, B. A double-hexamer archaeal minichromosome maintenance protein is an ATP-dependent DNA helicase. Proc. Natl. Acad. Sci. USA 97, 1530–1535 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Kelman, Z., Lee, J.K. & Hurwitz, J. The single minichromosome maintenance protein of Methanobacterium thermoautotrophicum DeltaH contains DNA helicase activity. Proc. Natl. Acad. Sci. USA 96, 14783–14788 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  33. Kanemaki, M., Sanchez-Diaz, A., Gambus, A. & Labib, K. Functional proteomic identification of DNA replication proteins by induced proteolysis in vivo. Nature 423, 720–724 (2003).

    Article  CAS  PubMed  Google Scholar 

  34. Takayama, Y. et al. GINS, a novel multiprotein complex required for chromosomal DNA replication in budding yeast. Genes Dev. 17, 1153–1165 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Boskovic, J. et al. Molecular architecture of the human GINS complex. EMBO Rep. 8, 678–684 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Choi, J.M., Lim, H.S., Kim, J.J., Song, O.K. & Cho, Y. Crystal structure of the human GINS complex. Genes Dev. 21, 1316–1321 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Chang, Y.P., Wang, G., Bermudez, V., Hurwitz, J. & Chen, X.S. Crystal structure of the GINS complex and functional insights into its role in DNA replication. Proc. Natl. Acad. Sci. USA 104, 12685–12690 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Kamada, K., Kubota, Y., Arata, T., Shindo, Y. & Hanaoka, F. Structure of the human GINS complex and its assembly and functional interface in replication initiation. Nat. Struct. Mol. Biol. 14, 388–396 (2007).

    Article  CAS  PubMed  Google Scholar 

  39. Grob, P. et al. Cryo-electron microscopy studies of human TFIID: conformational breathing in the integration of gene regulatory cues. Structure 14, 511–520 (2006).

    Article  CAS  PubMed  Google Scholar 

  40. van Heel, M., Harauz, G., Orlova, E.V., Schmidt, R. & Schatz, M. A new generation of the IMAGIC image processing system. J. Struct. Biol. 116, 17–24 (1996).

    Article  CAS  PubMed  Google Scholar 

  41. Im, J.S. et al. Assembly of the Cdc45-Mcm2–7-GINS complex in human cells requires the Ctf4/And-1, RecQL4, and Mcm10 proteins. Proc. Natl. Acad. Sci. USA 106, 15628–15632 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Makarova, K.S., Wolf, Y.I., Mekhedov, S.L., Mirkin, B.G. & Koonin, E.V. Ancestral paralogs and pseudoparalogs and their role in the emergence of the eukaryotic cell. Nucleic Acids Res. 33, 4626–4638 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Marinsek, N. et al. GINS, a central nexus in the archaeal DNA replication fork. EMBO Rep. 7, 539–545 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  44. Dean, F.B., Borowiec, J.A., Eki, T. & Hurwitz, J. The simian virus 40 T antigen double hexamer assembles around the DNA at the replication origin. J. Biol. Chem. 267, 14129–14137 (1992).

    CAS  PubMed  Google Scholar 

  45. Fouts, E.T., Yu, X., Egelman, E.H. & Botchan, M.R. Biochemical and electron microscopic image analysis of the hexameric E1 helicase. J. Biol. Chem. 274, 4447–4458 (1999).

    Article  CAS  PubMed  Google Scholar 

  46. 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  PubMed  Google Scholar 

  47. Labib, K. & Gambus, A. A key role for the GINS complex at DNA replication forks. Trends Cell Biol. 17, 271–278 (2007).

    Article  CAS  PubMed  Google Scholar 

  48. Takahashi, T.S., Wigley, D.B. & Walter, J.C. Pumps, paradoxes and ploughshares: mechanism of the MCM2–7 DNA helicase. Trends Biochem. Sci. 30, 437–444 (2005).

    Article  CAS  PubMed  Google Scholar 

  49. Takara, T.J. & Bell, S.P. Putting two heads together to unwind DNA. Cell 139, 652–654 (2009).

    Article  CAS  PubMed  Google Scholar 

  50. Yardimci, H., Loveland, A.B., Habuchi, S., van Oijen, A.M. & Walter, J.C. Uncoupling of sister replisomes during eukaryotic DNA replication. Mol. Cell 40, 834–840 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Gould, A.D. & Shilton, B.H. Studies of the maltose transport system reveal a mechanism for coupling ATP hydrolysis to substrate translocation without direct recognition of substrate. J. Biol. Chem. 285, 11290–11296 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Ludtke, S.J., Baldwin, P.R. & Chiu, W. EMAN: semiautomated software for high-resolution single-particle reconstructions. J. Struct. Biol. 128, 82–97 (1999).

    Article  CAS  PubMed  Google Scholar 

  53. Voss, N.R., Yoshioka, C.K., Radermacher, M., Potter, C.S. & Carragher, B. DoG Picker and TiltPicker: software tools to facilitate particle selection in single particle electron microscopy. J. Struct. Biol. 166, 205–213 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. van Heel, M. et al. Single-particle electron cryo-microscopy: towards atomic resolution. Q. Rev. Biophys. 33, 307–369 (2000).

    Article  CAS  PubMed  Google Scholar 

  55. Frank, J. et al. SPIDER and WEB: processing and visualization of images in 3D electron microscopy and related fields. J. Struct. Biol. 116, 190–199 (1996).

    Article  CAS  PubMed  Google Scholar 

  56. Boekema, E.J., Berden, J.A. & van Heel, M.G. Structure of mitochondrial F1-ATPase studied by electron microscopy and image processing. Biochim. Biophys. Acta 851, 353–360 (1986).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  58. Arnold, K., Bordoli, L., Kopp, J. & Schwede, T. The SWISS-MODEL workspace: a web-based environment for protein structure homology modelling. Bioinformatics 22, 195–201 (2006).

    Article  CAS  PubMed  Google Scholar 

  59. Krissinel, E. & Henrick, K. Secondary-structure matching (SSM), a new tool for fast protein structure alignment in three dimensions. Acta Crystallogr. D Biol. Crystallogr. 60, 2256–2268 (2004).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

The authors would like to thank A. Lyubimov and F. Bleichert for comments and help with the manuscript; and G. Lander, P. Grob, R. Hannah, R. Hall, M. Cianfrocco and C. Ciferri for technical help. This work was supported by a European Molecular Biology Organization long-term postdoctoral fellowship (to A.C.), a PhD fellowship from the Boehringer Ingelheim Fonds (to T.P.), the Human Frontier Science Program (RPG0039, to E.N.), the National Institute of General Medical Sciences (GM071747, to J.M.B.) and the National Cancer Institute (CA R37-30490, to M.R.B.). E.N. is a Howard Hughes Medical Institute investigator.

Author information

Authors and Affiliations

  1. Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, California, USA

    Alessandro Costa, Ivar Ilves, Nele Tamberg, Tatjana Petojevic, Eva Nogales, Michael R Botchan & James M Berger

  2. California Institute for Quantitative Biosciences, University of California, Berkeley, Berkeley, California, USA

    Alessandro Costa, Eva Nogales, Michael R Botchan & James M Berger

  3. Department of Biology, Chemistry and Pharmacy, Institute of Chemistry and Biochemistry, Freie Universität Berlin, Berlin, Germany

    Tatjana Petojevic

  4. Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, California, USA

    Eva Nogales

  5. Life Science Division, Lawrence Berkeley National Laboratory, Berkeley, California, USA

    Eva Nogales

Authors
  1. Alessandro Costa
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ivar Ilves
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Nele Tamberg
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Tatjana Petojevic
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Eva Nogales
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Michael R Botchan
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. James M Berger
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

A.C., I.I., M.R.B. and J.M.B. conceived the general ideas for this study. All authors planned experiments. A.C. did all electron microscopy single-particle reconstruction and molecular modeling supervised by J.M.B. and E.N. I.I., N.T. and T.P. did cloning, baculovirus construction and protein purification supervised by M.R.B. A.C., M.R.B. and J.M.B. wrote the manuscript. All authors provided editorial input.

Corresponding authors

Correspondence to Michael R Botchan or James M Berger.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–8 (PDF 1436 kb)

Supplementary Movie 1

The Mcm2-7 complex morphing between a planar-notched and a spiral-lockwasher configuration. (MOV 4475 kb)

Supplementary Movie 2

The CMG complex morphing between the apo and nucleotide-bound state (MOV 3815 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Costa, A., Ilves, I., Tamberg, N. et al. The structural basis for MCM2–7 helicase activation by GINS and Cdc45. Nat Struct Mol Biol 18, 471–477 (2011). https://doi.org/10.1038/nsmb.2004

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nsmb.2004

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