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Open-ringed structure of the Cdt1–Mcm2–7 complex as a precursor of the MCM double hexamer

Nature Structural & Molecular Biology volume 24, pages 300–308 (2017)Cite this article

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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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Figure 1: Overall structure of the heptamer saturated with AMPPNP.
Figure 2: Arrangement of N-terminal ZF and C-terminal WH motifs in the heptamer.
Figure 3: Interaction of Cdt1 with Mcm2–7.
Figure 4: Conformational changes of individual MCM subunits in the heptamer relative to the DH.
Figure 5: Structural comparison of the AMPPNP- or ADP-saturated heptamers and hexamers.
Figure 6: Models for loading and translocation of Mcm2–7 inspired by its coiled structure.

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References

  1. Zhu, W., Abbas, T. & Dutta, A. DNA replication and genomic instability. Adv. Exp. Med. Biol. 570, 249–279 (2005).

    Article  CAS  PubMed  Google Scholar 

  2. O'Donnell, M., Langston, L. & Stillman, B. Principles and concepts of DNA replication in bacteria, archaea, and eukarya. Cold Spring Harb. Perspect. Biol. 5, a010108 (2013).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  3. Bell, S.P. & Labib, K. Chromosome duplication in Saccharomyces cerevisiae. Genetics 203, 1027–1067 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Tanaka, S. & Diffley, J.F. Interdependent nuclear accumulation of budding yeast Cdt1 and Mcm2-7 during G1 phase. Nat. Cell Biol. 4, 198–207 (2002).

    Article  CAS  PubMed  Google Scholar 

  5. Tye, B.K. MCM proteins in DNA replication. Annu. Rev. Biochem. 68, 649–686 (1999).

    Article  CAS  PubMed  Google Scholar 

  6. Bochman, M.L. & Schwacha, A. The Mcm complex: unwinding the mechanism of a replicative helicase. Microbiol. Mol. Biol. Rev. 73, 652–683 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Bell, S.D. & Botchan, M.R. The minichromosome maintenance replicative helicase. Cold Spring Harb. Perspect. Biol. 5, a012807 (2013).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  8. Remus, D. et al. Concerted loading of Mcm2-7 double hexamers around DNA during DNA replication origin licensing. Cell 139, 719–730 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Evrin, C. et al. A double-hexameric MCM2-7 complex is loaded onto origin DNA during licensing of eukaryotic DNA replication. Proc. Natl. Acad. Sci. USA 106, 20240–20245 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Costa, A., Hood, I.V. & Berger, J.M. Mechanisms for initiating cellular DNA replication. Annu. Rev. Biochem. 82, 25–54 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Frigola, J., Remus, D., Mehanna, A. & Diffley, J.F. ATPase-dependent quality control of DNA replication origin licensing. Nature 495, 339–343 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Fernández-Cid, A. et al. An ORC/Cdc6/MCM2-7 complex is formed in a multistep reaction to serve as a platform for MCM double-hexamer assembly. Mol. Cell 50, 577–588 (2013).

    Article  PubMed  CAS  Google Scholar 

  13. Sun, J. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Ticau, S., Friedman, L.J., Ivica, N.A., Gelles, J. & Bell, S.P. Single-molecule studies of origin licensing reveal mechanisms ensuring bidirectional helicase loading. Cell 161, 513–525 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Sun, J. et al. Structural and mechanistic insights into Mcm2-7 double-hexamer assembly and function. Genes Dev. 28, 2291–2303 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  16. Li, N. et al. Structure of the eukaryotic MCM complex at 3.8 Å. Nature 524, 186–191 (2015).

    Article  CAS  PubMed  Google Scholar 

  17. Heller, R.C. et al. Eukaryotic origin-dependent DNA replication in vitro reveals sequential action of DDK and S-CDK kinases. Cell 146, 80–91 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Yeeles, J.T., Deegan, T.D., Janska, A., Early, A. & Diffley, J.F. Regulated eukaryotic DNA replication origin firing with purified proteins. Nature 519, 431–435 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Labib, K. How do Cdc7 and cyclin-dependent kinases trigger the initiation of chromosome replication in eukaryotic cells? Genes Dev. 24, 1208–1219 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Sheu, Y.J. & Stillman, B. Cdc7-Dbf4 phosphorylates MCM proteins via a docking site-mediated mechanism to promote S phase progression. Mol. Cell 24, 101–113 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Francis, L.I., Randell, J.C., Takara, T.J., Uchima, L. & Bell, S.P. Incorporation into the prereplicative complex activates the Mcm2-7 helicase for Cdc7-Dbf4 phosphorylation. Genes Dev. 23, 643–654 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Deegan, T.D., Yeeles, J.T. & Diffley, J.F. Phosphopeptide binding by Sld3 links Dbf4-dependent kinase to MCM replicative helicase activation. EMBO J. 35, 961–973 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Tanaka, S., Nakato, R., Katou, Y., Shirahige, K. & Araki, H. Origin association of Sld3, Sld7, and Cdc45 proteins is a key step for determination of origin-firing timing. Curr. Biol. 21, 2055–2063 (2011).

    Article  CAS  PubMed  Google Scholar 

  24. Zegerman, P. & Diffley, J.F. Phosphorylation of Sld2 and Sld3 by cyclin-dependent kinases promotes DNA replication in budding yeast. Nature 445, 281–285 (2007).

    Article  CAS  PubMed  Google Scholar 

  25. Tanaka, S. et al. CDK-dependent phosphorylation of Sld2 and Sld3 initiates DNA replication in budding yeast. Nature 445, 328–332 (2007).

    Article  CAS  PubMed  Google Scholar 

  26. Tanaka, S. & Araki, H. Helicase activation and establishment of replication forks at chromosomal origins of replication. Cold Spring Harb. Perspect. Biol. 5, a010371 (2013).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  27. Muramatsu, S., Hirai, K., Tak, Y.S., Kamimura, Y. & Araki, H. CDK-dependent complex formation between replication proteins Dpb11, Sld2, Pol (epsilon, and GINS in budding yeast. Genes Dev. 24, 602–612 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Fu, Y.V. et al. Selective bypass of a lagging strand roadblock by the eukaryotic replicative DNA helicase. Cell 146, 931–941 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Samel, S.A. et al. A unique DNA entry gate serves for regulated loading of the eukaryotic replicative helicase MCM2-7 onto DNA. Genes Dev. 28, 1653–1666 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Takara, T.J. & Bell, S.P. Multiple Cdt1 molecules act at each origin to load replication-competent Mcm2-7 helicases. EMBO J. 30, 4885–4896 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Riera, A., Tognetti, S. & Speck, C. Helicase loading: how to build a MCM2-7 double-hexamer. Semin. Cell Dev. Biol. 30, 104–109 (2014).

    Article  CAS  PubMed  Google Scholar 

  32. Coster, G., Frigola, J., Beuron, F., Morris, E.P. & Diffley, J.F. Origin licensing requires ATP binding and hydrolysis by the MCM replicative helicase. Mol. Cell 55, 666–677 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Kang, S., Warner, M.D. & Bell, S.P. Multiple functions for Mcm2-7 ATPase motifs during replication initiation. Mol. Cell 55, 655–665 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Miller, J.M. & Enemark, E.J. Archaeal MCM proteins as an analog for the eukaryotic Mcm2-7 helicase to reveal essential features of structure and function. Archaea 2015, 305497 (2015).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  35. Chong, J.P., Hayashi, M.K., Simon, M.N., Xu, R.M. & Stillman, B. A double-hexamer archaeal minichromosome maintenance protein is an ATP-dependent DNA helicase. Proc. Natl. Acad. Sci. USA 97, 1530–1535 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Slaymaker, I.M. & Chen, X.S. MCM structure and mechanics: what we have learned from archaeal MCM. Subcell. Biochem. 62, 89–111 (2012).

    Article  CAS  PubMed  Google Scholar 

  37. Lyubimov, A.Y., Costa, A., Bleichert, F., Botchan, M.R. & Berger, J.M. ATP-dependent conformational dynamics underlie the functional asymmetry of the replicative helicase from a minimalist eukaryote. Proc. Natl. Acad. Sci. USA 109, 11999–12004 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Costa, A. et al. The structural basis for MCM2-7 helicase activation by GINS and Cdc45. Nat. Struct. Mol. Biol. 18, 471–477 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Yuan, Z. et al. Structure of the eukaryotic replicative CMG helicase suggests a pumpjack motion for translocation. Nat. Struct. Mol. Biol. 23, 217–224 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Wu, R., Wang, J. & Liang, C. Cdt1p, through its interaction with Mcm6p, is required for the formation, nuclear accumulation and chromatin loading of the MCM complex. J. Cell Sci. 125, 209–219 (2012).

    Article  CAS  PubMed  Google Scholar 

  41. Chen, S. & Bell, S.P. CDK prevents Mcm2-7 helicase loading by inhibiting Cdt1 interaction with Orc6. Genes Dev. 25, 363–372 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Liu, C. et al. Structural insights into the Cdt1-mediated MCM2-7 chromatin loading. Nucleic Acids Res. 40, 3208–3217 (2012).

    Article  CAS  PubMed  Google Scholar 

  43. Khayrutdinov, B.I. et al. Structure of the Cdt1 C-terminal domain: conservation of the winged helix fold in replication licensing factors. Protein Sci. 18, 2252–2264 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Evrin, C. et al. The ORC/Cdc6/MCM2-7 complex facilitates MCM2-7 dimerization during prereplicative complex formation. Nucleic Acids Res. 42, 2257–2269 (2014).

    Article  CAS  PubMed  Google Scholar 

  45. Bochman, M.L. & Schwacha, A. The Mcm2-7 complex has in vitro helicase activity. Mol. Cell 31, 287–293 (2008).

    Article  CAS  PubMed  Google Scholar 

  46. Miller, J.M., Arachea, B.T., Epling, L.B. & Enemark, E.J. Analysis of the crystal structure of an active MCM hexamer. eLife 3, e03433 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  47. Froelich, C.A., Kang, S., Epling, L.B., Bell, S.P. & Enemark, E.J. A conserved MCM single-stranded DNA binding element is essential for replication initiation. eLife 3, e01993 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  48. Enemark, E.J. & Joshua-Tor, L. On helicases and other motor proteins. Curr. Opin. Struct. Biol. 18, 243–257 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Lyubimov, A.Y., Strycharska, M. & Berger, J.M. The nuts and bolts of ring-translocase structure and mechanism. Curr. Opin. Struct. Biol. 21, 240–248 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. O'Shea, V.L. & Berger, J.M. Loading strategies of ring-shaped nucleic acid translocases and helicases. Curr. Opin. Struct. Biol. 25, 16–24 (2014).

    Article  CAS  PubMed  Google Scholar 

  51. Arias-Palomo, E., O'Shea, V.L., Hood, I.V. & Berger, J.M. The bacterial DnaC helicase loader is a DnaB ring breaker. Cell 153, 438–448 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Samson, R.Y., Abeyrathne, P.D. & Bell, S.D. Mechanism of archaeal MCM helicase recruitment to DNA replication origins. Mol. Cell 61, 287–296 (2016).

    Article  CAS  PubMed  Google Scholar 

  53. Bowman, G.D., O'Donnell, M. & Kuriyan, J. Structural analysis of a eukaryotic sliding DNA clamp-clamp loader complex. Nature 429, 724–730 (2004).

    Article  CAS  PubMed  Google Scholar 

  54. Yao, N.Y. & O'Donnell, M. The RFC clamp loader: structure and function. Subcell. Biochem. 62, 259–279 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Abid Ali, F. et al. Cryo-EM structures of the eukaryotic replicative helicase bound to a translocation substrate. Nat. Commun. 7, 10708 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Chen, S., de Vries, M.A. & Bell, S.P. Orc6 is required for dynamic recruitment of Cdt1 during repeated Mcm2-7 loading. Genes Dev. 21, 2897–2907 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Bowers, J.L., Randell, J.C., Chen, S. & Bell, S.P. ATP hydrolysis by ORC catalyzes reiterative Mcm2-7 assembly at a defined origin of replication. Mol. Cell 16, 967–978 (2004).

    Article  CAS  PubMed  Google Scholar 

  58. Costa, A. et al. DNA binding polarity, dimerization, and ATPase ring remodeling in the CMG helicase of the eukaryotic replisome. eLife 3, e03273 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  59. Syed, S., Pandey, M., Patel, S.S. & Ha, T. Single-molecule fluorescence reveals the unwinding stepping mechanism of replicative helicase. Cell Rep. 6, 1037–1045 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Itsathitphaisarn, O., Wing, R.A., Eliason, W.K., Wang, J. & Steitz, T.A. The hexameric helicase DnaB adopts a nonplanar conformation during translocation. Cell 151, 267–277 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  61. Zhao, M. et al. Mechanistic insights into the recycling machine of the SNARE complex. Nature 518, 61–67 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  62. Li, X. et al. Electron counting and beam-induced motion correction enable near-atomic-resolution single-particle cryo-EM. Nat. Methods 10, 584–590 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  63. Shaikh, T.R. et al. SPIDER image processing for single-particle reconstruction of biological macromolecules from electron micrographs. Nat. Protoc. 3, 1941–1974 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  64. Scheres, S.H. RELION: implementation of a Bayesian approach to cryo-EM structure determination. J. Struct. Biol. 180, 519–530 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  65. Mindell, J.A. & Grigorieff, N. Accurate determination of local defocus and specimen tilt in electron microscopy. J. Struct. Biol. 142, 334–347 (2003).

    Article  PubMed  Google Scholar 

  66. Pettersen, E.F. et al. UCSF Chimera—a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004).

    Article  CAS  PubMed  Google Scholar 

  67. Buchan, D.W., Minneci, F., Nugent, T.C., Bryson, K. & Jones, D.T. Scalable web services for the PSIPRED Protein Analysis Workbench. Nucleic Acids Res. 41, W349–W357 (2013).

    Article  PubMed  PubMed Central  Google Scholar 

  68. Yang, J. et al. The I-TASSER Suite: protein structure and function prediction. Nat. Methods 12, 7–8 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  69. Lee, C. et al. Structural basis for inhibition of the replication licensing factor Cdt1 by geminin. Nature 430, 913–917 (2004).

    Article  CAS  PubMed  Google Scholar 

  70. De Marco, V. et al. Quaternary structure of the human Cdt1-Geminin complex regulates DNA replication licensing. Proc. Natl. Acad. Sci. USA 106, 19807–19812 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  71. Jee, J. et al. Structure and mutagenesis studies of the C-terminal region of licensing factor Cdt1 enable the identification of key residues for binding to replicative helicase Mcm proteins. J. Biol. Chem. 285, 15931–15940 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  72. Wiedemann, C. et al. Structure and regulatory role of the C-terminal winged helix domain of the archaeal minichromosome maintenance complex. Nucleic Acids Res. 43, 2958–2967 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  73. Afonine, P.V. et al. Towards automated crystallographic structure refinement with phenix.refine. Acta Crystallogr. D Biol. Crystallogr. 68, 352–367 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  74. Adams, P.D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D Biol. Crystallogr. 66, 213–221 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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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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Author notes

  1. Yuanliang Zhai and Erchao Cheng: These authors contributed equally to this work.

Authors and Affiliations

  1. Division of Life Science, Hong Kong University of Science and Technology, Clear Water Bay, Hong Kong, China

    Yuanliang Zhai, Philip Yuk Kwong Yung & Bik-Kwoon Tye

  2. Institute for Advanced Study, Hong Kong University of Science and Technology, Clear Water Bay, Hong Kong, China

    Yuanliang Zhai

  3. Ministry of Education Key Laboratory of Protein Sciences, Beijing Advanced Innovation Center for Structural Biology, School of Life Sciences, Tsinghua University, Beijing, China

    Erchao Cheng, Hao Wu & Ning Gao

  4. Peking-Tsinghua Center for Life Sciences, School of Life Sciences, Peking University, Beijing, China

    Ningning Li

  5. Department of Molecular Biology and Genetics, College of Agriculture and Life Sciences, Cornell University, Ithaca, New York, USA

    Bik-Kwoon Tye

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Contributions

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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Correspondence to Ning Gao or Bik-Kwoon Tye.

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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).

Supplementary information

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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