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


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.

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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.

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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




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.

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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)

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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).

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