Abstract
The minichromosome maintenance complex (MCM) hexameric complex (Mcm2–7) forms the core of the eukaryotic replicative helicase. During G1 phase, two Cdt1–Mcm2–7 heptamers are loaded onto each replication origin by the origin-recognition complex (ORC) and Cdc6 to form an inactive MCM double hexamer (DH), but the detailed loading mechanism remains unclear. Here we examine the structures of the yeast MCM hexamer and Cdt1–MCM heptamer from Saccharomyces cerevisiae. Both complexes form left-handed coil structures with a 10–15-Å gap between Mcm5 and Mcm2, and a central channel that is occluded by the C-terminal domain winged-helix motif of Mcm5. Cdt1 wraps around the N-terminal regions of Mcm2, Mcm6 and Mcm4 to stabilize the whole complex. The intrinsic coiled structures of the precursors provide insights into the DH formation, and suggest a spring-action model for the MCM during the initial origin melting and the subsequent DNA unwinding.
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Acknowledgements
We thank the Tsinghua University Branch of the China National Center for Protein Sciences (Beijing, China) for technical support with cryo-EM data collection and for computation resources. Part of the computation was done on the Computing Platform of the Center for Life Science, Peking University. We also thank J. Diffley and S. Gasser (Francis Crick Institute, London, UK) for yeast and E. coli strains. This work was supported by the Ministry of Science and Technology of China (grants 2013CB910404 and 2016YFA0500700 to N.G.), the National Natural Science Foundation of China (grants 31422016, 31470722 and 31630087 to N.G.), the Research Grants Council of Hong Kong (grants GRF16138716 to B.-K.T.; GRF664013, HKUST12/CRF/13G, GRF16104115 and GRF16143016 to Y.Z. and B.-K.T.; and IGN15SC02 to Y.Z.). N.L. is supported by a postdoctoral fellowship from the Peking-Tsinghua Center for Life Sciences.
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Y.Z. and P.Y.K.Y. purified proteins; E.C., H.W. and N.L. collected and processed data; P.Y.K.Y., Y.Z. and E.C. generated animations; and Y.Z., E.C., N.G. and B.-K.T. prepared the manuscript.
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Integrated supplementary information
Supplementary Figure 1 Purification, SDS-PAGE and cryo-EM characterization of the MCM hexamer and Cdt1–MCM heptamer samples.
(a-b) SDS-PAGE analysis of the hexamer and heptamer samples. The eluted hexamer (a) and heptamer (b) complexes were subjected to 20–40% glycerol gradient sedimentation centrifugation. Collected fractions were analyzed by SDS-PAGE and visualized by coomassie blue staining. Molecular size markers used are: ALP 140 kDa and thyroglobulin 669 kDa. Fractions 7-9 (a) and 8-10 (b) were pooled and concentrated for cryo-EM analysis. (c) The sedimentation profile of Mcm5 and Cdt1 from the hexamer (a) and heptamer (b) samples were plotted using quantified signals by Image J. (d) A representative raw micrograph of Cdt1–Mcm2-7 complexes in vitreous ice. (e-g) Representative 2D class averages of cryo-EM particles from the datasets of heptamer+AMPPNP (e), heptamer+ADP (f), and hexamer+ADP+NP40 (g). See also Table 1.
Supplementary Figure 2 Flowchart for image processing of particles from the heptamer + AMPPNP data set and Fourier shell correlation curves of the five structures.
(a) A flowchart of the steps in image processing. (b-f), Fourier Shell Correlation curves of the heptamer–AMPPNP (b), heptamer–ADP (c), hexamer–AMPPNP (d), hexamer–ADP (e) and (hexamer–ADP)* (f) structures. Note that (hexamer–ADP)* is the hexamer structure from the dataset of heptamer + ADP. See also Table 1.
Supplementary Figure 3 Overall structures of the heptamer and hexamer.
(a-c) Top panels, The cryo-EM maps of the indicated complexes are shown in surface representation, with individual components color coded. Bottom panel, the maps are shown in transparent surface representation, with models superimposed. Both the CTD top-view and consecutively rotated side-views are shown. a, hexamer–AMPPNP. b, heptamer–ADP c, hexamer–ADP.
Supplementary Figure 4 Arrangements of oligonucleic-acid-binding subdomains (OBs) in the heptamer and double hexamer.
(a-b) Arrangements of OB subdomains from the heptamer (a), and the double hexamer (b). Models are shown in ribbon representation. Individual OB subdomains are color-coded. (c-d) Superimposition of OB subdomains from heptamer (color-coded) and double hexamer (grey), shown in top (c) and side (d) views. The alignment is done using the OB of Mcm7 as reference.
Supplementary Figure 5 Constellation of Cdt1-binding sites in the heptamer and double hexamer.
(a) The binding sites of Cdt1 on the NTD of Mcm2 (orange), CTD-A of Mcm6 (blue) and NTD of Mcm4 (magenta) as well as the two interacting N-C-linkers of Mcm6 (sky blue) and Mcm4 (red) in the heptamer are highlighted in different colors. (b) The MCM single hexamer from the double hexamer is aligned to the heptamer and shown in a similar orientation as in (a), with Cdt1 binding sites mapped. The NTD of Mcm6 (tan) is used as reference for the alignment.
Supplementary Figure 6 Structural comparison of the heptamer and double hexamer.
(a-f) Conformational differences of each NTDs from the heptamer and the double hexamer. NTD-A was used as reference for the alignment. NTDs of the heptamer are color coded and the corresponding NTDs in the DH are in grey. Dramatic changes are observed for the ZF motifs of Mcm2 (20 Å) and Mcm3 (10 Å). The distance changes of the other motifs of NTDs (ZF and OB) are measured by r.m.s.d. (root-mean-square deviation). r.m.s.d. values: M2 (7.5 Å), M6 (1.9 Å), M4 (2.0 Å), M7 (1.6 Å), M3 (3.5 Å), M5 (2.2 Å). (g-k) Conformational differences of the CTD-A dimers in the heptamer (color-coded for subunits) and in the double hexamer (grey). One CTD-A of each neighboring pairs (the left one) was used as the reference for alignment. Calculated r.m.s.d values of the other CTD-As are: M6 (4.3 Å), M4 (6.0 Å), M7 (6.8 Å), M3 (6.1 Å), M5 (5.0 Å).
Supplementary Figure 7 Cdc45 and GINS are structurally incompatible for interactions with MCM subunits in the heptamer and open- and closed-ring conformations of different MCM complexes.
(a) Structure of the CMG complex in surface representation. The map of the CMG was converted from a previous atomic model (PDB 3JC5). Mcm5, Mcm2, GINS, Cdc45 are colored yellow, orange, sky blue and purple, respectively. (b) Structure of the Cdt1–Mcm2-7 heptamer, displayed in a comparable orientation as in (a). (c-d) Superimposition of Cdc45 and GINS from the CMG structure onto the map of the heptamer, showing in top (c) and side (d) views. Mcm2-NTD was used as reference for the alignment. As shown, aligned Cdc45/GINS exhibits structural conflict with the CTD-A of Mcm5 in the heptamer, indicating that the interactions of Cdc45/GINS with the NTDs of Mcm5 and Mcm2 observed in the CMG complex could not be simultaneously satisfied in the heptamer. (e) CTD top views of the cryo-EM maps (grey) of the yeast MCM hexamer, Cdt1–MCM heptamer, and double hexamer (EMD-6338). The double hexamer map is low-pass filter to 10 Å. (f) CTD top views of the two conformers of the yeast CMG complex (EMD-6536 and EMD-6535). (g) CTD top views of the two conformers of the Drosophila CMG complex (EMD-3321 and EMD-3320).
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Supplementary Text and Figures
Supplementary Figures 1–7 (PDF 1596 kb)
Supplementary Video 1
Structure of the Cdt1-Mcm2-7 complex and its transition to the MCM double hexamer. The cryo-EM map of the Cdt1-MCM heptamer (AMPPNP saturated state) is shown in transparent surface representation, superimposed with color-coded subunits and Cdt1. After a continuous rotation around the channel axis, the open-ring conformation of the heptamer morphs into the closed ring conformation of the double hexamer. (MP4 13719 kb)
Supplementary Video 2
Proposed spring-action model for the translocation of the MCM complex on DNA. The MCM core of the CMG helicase, which translocates on DNA via a spring-action mechanism, is illustrated by animation. The repeated spring action of transitioning between the open- and closed-ring conformations translocates the helicase in an inchworm-like motion along one strand of the vertically displayed duplex DNA from bottom to top. The gap-forming Mcm5 and Mcm2 are labeled. (MP4 6334 kb)
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Zhai, Y., Cheng, E., Wu, H. et al. Open-ringed structure of the Cdt1–Mcm2–7 complex as a precursor of the MCM double hexamer. Nat Struct Mol Biol 24, 300–308 (2017). https://doi.org/10.1038/nsmb.3374
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DOI: https://doi.org/10.1038/nsmb.3374
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