Article | Published:

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

Nature Structural & Molecular Biology volume 18, pages 471477 (2011) | Download Citation

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.

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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

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

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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.

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The authors declare no competing financial interests.

Corresponding authors

Correspondence to Michael R Botchan or James M Berger.

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–8

Videos

  1. 1.

    Supplementary Movie 1

    The Mcm2-7 complex morphing between a planar-notched and a spiral-lockwasher configuration.

  2. 2.

    Supplementary Movie 2

    The CMG complex morphing between the apo and nucleotide-bound state

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DOI

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

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