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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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.
The authors declare no competing financial interests.
Supplementary Figures 1–5 (PDF 675 kb)
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)
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). https://doi.org/10.1038/nsmb.2629
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