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Structure of the eukaryotic replicative CMG helicase suggests a pumpjack motion for translocation

Nature Structural & Molecular Biology volume 23, pages 217224 (2016) | Download Citation

Abstract

The CMG helicase is composed of Cdc45, Mcm2–7 and GINS. Here we report the structure of the Saccharomyces cerevisiae CMG, determined by cryo-EM at a resolution of 3.7–4.8 Å. The structure reveals that GINS and Cdc45 scaffold the N tier of the helicase while enabling motion of the AAA+ C tier. CMG exists in two alternating conformations, compact and extended, thus suggesting that the helicase moves like an inchworm. The N-terminal regions of Mcm2–7, braced by Cdc45–GINS, form a rigid platform upon which the AAA+ C domains make longitudinal motions, nodding up and down like an oil-rig pumpjack attached to a stable platform. The Mcm ring is remodeled in CMG relative to the inactive Mcm2–7 double hexamer. The Mcm5 winged-helix domain is inserted into the central channel, thus blocking entry of double-stranded DNA and supporting a steric-exclusion DNA-unwinding model.

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Acknowledgements

Cryo-EM data were collected on a Titan Krios I at the Howard Hughes Medical Institute, Janelia Farm. We also collected a cryo-EM data set on an FEI Polara with a K2 detector at the University of Texas Health Science Center. We thank the staff at these facilities for help with data collection. We also thank L. Pellegrini (University of Cambridge) for sharing the structure of human Cdc45 before publication. This work was funded by the US National Institutes of Health (GM111472 and OD12272 to H.L. and GM115809 to M.E.O'D.) and the Howard Hughes Medical Institute (M.E.O'D.).

Author information

Affiliations

  1. Department of Biochemistry & Cell Biology, Stony Brook University, Stony Brook, New York, USA.

    • Zuanning Yuan
    •  & Huilin Li
  2. Biology Department, Brookhaven National Laboratory, Upton, New York, USA.

    • Zuanning Yuan
    • , Lin Bai
    • , Jingchuan Sun
    •  & Huilin Li
  3. DNA Replication Laboratory, Rockefeller University, New York, New York, USA.

    • Roxana Georgescu
    •  & Michael E O'Donnell
  4. Howard Hughes Medical Institute, Rockefeller University, New York, New York, USA.

    • Roxana Georgescu
    •  & Michael E O'Donnell
  5. Department of Pathology and Laboratory Medicine, University of Texas Medical School at Houston, Houston, Texas, USA.

    • Jun Liu

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Contributions

Z.Y., L.B., J.S., R.G., M.E.O'D. and H.L. designed experiments. Z.Y., L.B., J.S., R.G. and J.L. performed experiments. Z.Y., L.B., J.S. and H.L. analyzed the data. M.E.O'D. and H.L. wrote the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Michael E O'Donnell or Huilin Li.

Integrated supplementary information

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–7 and Supplementary Table 1

Videos

  1. 1.

    Morphing of the CMG structure between conformers I and II

    The video starts with an N-face view of conformer I, rotates it 90o to a back side view, then rotates it 90o to a front side view and morphs conformer I with conformer II. Then CMG conformer I is rotated 90o to a side view in which the GINSCdc45 project toward the viewer, and morphs it with conformer II.

  2. 2.

    Morphing of the Mcm 5,2,6,4 subunits of CMG, showing major changes between conformers I and II

    The Mcm5,2,6,4 subunits are colored yellow, blue, red, and green, respectively, and other subunits of CMG are removed for clarity.

  3. 3.

    Animation of pumpjack translocation

    An animation illustrating the proposed DNA unwinding and translocation mechanism of the CMG helicase, showing the side view of a ratcheting CMG acting as a nodding pumpjack while traveling from left to right on a horizontal DNA.

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DOI

https://doi.org/10.1038/nsmb.3170

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