Structural basis of Mcm2–7 replicative helicase loading by ORC–Cdc6 and Cdt1


To initiate DNA replication, the origin recognition complex (ORC) and Cdc6 load an Mcm2–7 double hexamer onto DNA. Without ATP hydrolysis, ORC–Cdc6 recruits one Cdt1-bound Mcm2–7 hexamer, thus forming an ORC–Cdc6–Cdt1–Mcm2–7 (OCCM) helicase-loading intermediate. Here we report a 3.9-Å structure of Saccharomyces cerevisiae OCCM on DNA. Flexible Mcm2–7 winged-helix domains (WHDs) engage ORC–Cdc6. A three-domain Cdt1 configuration embraces Mcm2, Mcm4, and Mcm6, thus comprising nearly half of the hexamer. The Cdt1 C-terminal domain extends to the Mcm6 WHD, which binds the Orc4 WHD. DNA passes through the ORC–Cdc6 and Mcm2–7 rings. Origin DNA interaction is mediated by an α-helix within Orc4 and positively charged loops within Orc2 and Cdc6. The Mcm2–7 C-tier AAA+ ring is topologically closed by an Mcm5 loop that embraces Mcm2, but the N-tier-ring Mcm2-Mcm5 interface remains open. This structure suggests a loading mechanism of the first Cdt1-bound Mcm2–7 hexamer by ORC–Cdc6.

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Figure 1: Cryo-EM images and overall structure of the S. cerevisiae OCCM complex.
Figure 2: CLMS analysis of the S. cerevisiae OCCM complex.
Figure 3: ORC–Cdc6 encircles the origin DNA, and the Orc4 insertion helix binds to the major groove.
Figure 4: Nucleotide-binding sites and configuration in OCCM.
Figure 5: Extensive interactions between Cdt1 and the Mcm hexamer.
Figure 6: Mcm2–7 conformational changes between OCCM and the DH.
Figure 7: Interactions between OCCM and DNA.

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  1. 1

    Bell, S.P. & Dutta, A. DNA replication in eukaryotic cells. Annu. Rev. Biochem. 71, 333–374 (2002).

    CAS  PubMed  Google Scholar 

  2. 2

    Bell, S.P. & Stillman, B. ATP-dependent recognition of eukaryotic origins of DNA replication by a multiprotein complex. Nature 357, 128–134 (1992).

    CAS  PubMed  PubMed Central  Google Scholar 

  3. 3

    Stillman, B. Origin recognition and the chromosome cycle. FEBS Lett. 579, 877–884 (2005).

    CAS  PubMed  Google Scholar 

  4. 4

    Li, H. & Stillman, B. The origin recognition complex: a biochemical and structural view. Subcell. Biochem. 62, 37–58 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  5. 5

    Chen, Z. et al. The architecture of the DNA replication origin recognition complex in Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA 105, 10326–10331 (2008).

    CAS  PubMed  Google Scholar 

  6. 6

    Sun, J. et al. Cdc6-induced conformational changes in ORC bound to origin DNA revealed by cryo-electron microscopy. Structure 20, 534–544 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  7. 7

    Bleichert, F., Botchan, M.R. & Berger, J.M. Crystal structure of the eukaryotic origin recognition complex. Nature 519, 321–326 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  8. 8

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

    CAS  PubMed  PubMed Central  Google Scholar 

  9. 9

    Speck, C., Chen, Z., Li, H. & Stillman, B. ATPase-dependent cooperative binding of ORC and Cdc6 to origin DNA. Nat. Struct. Mol. Biol. 12, 965–971 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  10. 10

    Speck, C. & Stillman, B. Cdc6 ATPase activity regulates ORC × Cdc6 stability and the selection of specific DNA sequences as origins of DNA replication. J. Biol. Chem. 282, 11705–11714 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. 11

    Cocker, J.H., Piatti, S., Santocanale, C., Nasmyth, K. & Diffley, J.F. An essential role for the Cdc6 protein in forming the pre-replicative complexes of budding yeast. Nature 379, 180–182 (1996).

    CAS  PubMed  Google Scholar 

  12. 12

    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).

    CAS  PubMed  Google Scholar 

  13. 13

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

    CAS  PubMed  PubMed Central  Google Scholar 

  14. 14

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

    CAS  Google Scholar 

  15. 15

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

    PubMed  PubMed Central  Google Scholar 

  16. 16

    Moyer, S.E., Lewis, P.W. & Botchan, M.R. Isolation of the Cdc45/Mcm2-7/GINS (CMG) complex, a candidate for the eukaryotic DNA replication fork helicase. Proc. Natl. Acad. Sci. USA 103, 10236–10241 (2006).

    CAS  PubMed  Google Scholar 

  17. 17

    Gambus, A. et al. GINS maintains association of Cdc45 with MCM in replisome progression complexes at eukaryotic DNA replication forks. Nat. Cell Biol. 8, 358–366 (2006).

    CAS  PubMed  Google Scholar 

  18. 18

    Remus, D. & Diffley, J.F. Eukaryotic DNA replication control: lock and load, then fire. Curr. Opin. Cell Biol. 21, 771–777 (2009).

    CAS  PubMed  Google Scholar 

  19. 19

    Yardimci, H., Loveland, A.B., Habuchi, S., van Oijen, A.M. & Walter, J.C. Uncoupling of sister replisomes during eukaryotic DNA replication. Mol. Cell 40, 834–840 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  20. 20

    Botchan, M. & Berger, J. DNA replication: making two forks from one prereplication complex. Mol. Cell 40, 860–861 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  21. 21

    Aparicio, T., Guillou, E., Coloma, J., Montoya, G. & Méndez, J. The human GINS complex associates with Cdc45 and MCM and is essential for DNA replication. Nucleic Acids Res. 37, 2087–2095 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. 22

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

    CAS  PubMed  PubMed Central  Google Scholar 

  23. 23

    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).

    CAS  PubMed  Google Scholar 

  24. 24

    Araki, H. Cyclin-dependent kinase-dependent initiation of chromosomal DNA replication. Curr. Opin. Cell Biol. 22, 766–771 (2010).

    CAS  PubMed  Google Scholar 

  25. 25

    Sheu, Y.J. & Stillman, B. The Dbf4-Cdc7 kinase promotes S phase by alleviating an inhibitory activity in Mcm4. Nature 463, 113–117 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  26. 26

    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).

    CAS  PubMed  PubMed Central  Google Scholar 

  27. 27

    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).

    PubMed  PubMed Central  Google Scholar 

  28. 28

    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).

    CAS  PubMed  PubMed Central  Google Scholar 

  29. 29

    Evrin, C. et al. In the absence of ATPase activity, pre-RC formation is blocked prior to MCM2-7 hexamer dimerization. Nucleic Acids Res. 41, 3162–3172 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. 30

    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).

    CAS  PubMed  PubMed Central  Google Scholar 

  31. 31

    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).

    CAS  PubMed  PubMed Central  Google Scholar 

  32. 32

    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).

    PubMed  Google Scholar 

  33. 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).

    CAS  PubMed  PubMed Central  Google Scholar 

  34. 34

    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).

    CAS  PubMed  PubMed Central  Google Scholar 

  35. 35

    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).

    CAS  PubMed  Google Scholar 

  36. 36

    Chang, F. et al. Cdc6 ATPase activity disengages Cdc6 from the pre-replicative complex to promote DNA replication. eLife 4, e05795 (2015).

    PubMed Central  Google Scholar 

  37. 37

    Bleichert, F. et al. A Meier-Gorlin syndrome mutation in a conserved C-terminal helix of Orc6 impedes origin recognition complex formation. eLife 2, e00882 (2013).

    PubMed  PubMed Central  Google Scholar 

  38. 38

    Balasov, M., Akhmetova, K. & Chesnokov, I. Drosophila model of Meier-Gorlin syndrome based on the mutation in a conserved C-terminal domain of Orc6. Am. J. Med. Genet. A. 167A, 2533–2540 (2015).

    PubMed  PubMed Central  Google Scholar 

  39. 39

    Gaudier, M., Schuwirth, B.S., Westcott, S.L. & Wigley, D.B. Structural basis of DNA replication origin recognition by an ORC protein. Science 317, 1213–1216 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  40. 40

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

    CAS  PubMed  Google Scholar 

  41. 41

    Wei, Z. et al. Characterization and structure determination of the Cdt1 binding domain of human minichromosome maintenance (Mcm) 6. J. Biol. Chem. 285, 12469–12473 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  42. 42

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

    CAS  PubMed  Google Scholar 

  43. 43

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

    CAS  PubMed  Google Scholar 

  44. 44

    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).

    CAS  PubMed  PubMed Central  Google Scholar 

  45. 45

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

    CAS  PubMed  PubMed Central  Google Scholar 

  46. 46

    Neuwald, A.F., Aravind, L., Spouge, J.L. & Koonin, E.V. AAA+: a class of chaperone-like ATPases associated with the assembly, operation, and disassembly of protein complexes. Genome Res. 9, 27–43 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  47. 47

    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).

    CAS  PubMed  PubMed Central  Google Scholar 

  48. 48

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

    CAS  PubMed  PubMed Central  Google Scholar 

  49. 49

    O'Donnell, M. & Kuriyan, J. Clamp loaders and replication initiation. Curr. Opin. Struct. Biol. 16, 35–41 (2006).

    CAS  PubMed  Google Scholar 

  50. 50

    Kelch, B.A., Makino, D.L., O'Donnell, M. & Kuriyan, J. How a DNA polymerase clamp loader opens a sliding clamp. Science 334, 1675–1680 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  51. 51

    Dueber, E.L.C., Corn, J.E., Bell, S.D. & Berger, J.M. Replication origin recognition and deformation by a heterodimeric archaeal Orc1 complex. Science 317, 1210–1213 (2007).

    CAS  PubMed  Google Scholar 

  52. 52

    Suck, D. & Oefner, C. Structure of DNase I at 2.0 A resolution suggests a mechanism for binding to and cutting DNA. Nature 321, 620–625 (1986).

    CAS  PubMed  Google Scholar 

  53. 53

    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).

    CAS  PubMed  PubMed Central  Google Scholar 

  54. 54

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

    CAS  PubMed  PubMed Central  Google Scholar 

  55. 55

    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).

    CAS  PubMed  Google Scholar 

  56. 56

    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).

    CAS  PubMed  PubMed Central  Google Scholar 

  57. 57

    Rohou, A. & Grigorieff, N. CTFFIND4: fast and accurate defocus estimation from electron micrographs. J. Struct. Biol. 192, 216–221 (2015).

    PubMed  PubMed Central  Google Scholar 

  58. 58

    Scheres, S.H. Semi-automated selection of cryo-EM particles in RELION-1.3. J. Struct. Biol. 189, 114–122 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  59. 59

    Biasini, M. et al. SWISS-MODEL: modelling protein tertiary and quaternary structure using evolutionary information. Nucleic Acids Res. 42, W252–W258 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  60. 60

    Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 60, 2126–2132 (2004).

    Google Scholar 

  61. 61

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

    CAS  Google Scholar 

  62. 62

    Li, W., Zhang, T. & Ding, J. Molecular basis for the substrate specificity and catalytic mechanism of thymine-7-hydroxylase in fungi. Nucleic Acids Res. 43, 10026–10038 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  63. 63

    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).

    CAS  PubMed  Google Scholar 

  64. 64

    Doyle, J.M., Gao, J., Wang, J., Yang, M. & Potts, P.R. MAGE-RING protein complexes comprise a family of E3 ubiquitin ligases. Mol. Cell 39, 963–974 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  65. 65

    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).

    CAS  PubMed  PubMed Central  Google Scholar 

  66. 66

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

    CAS  PubMed  PubMed Central  Google Scholar 

  67. 67

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

    CAS  PubMed  PubMed Central  Google Scholar 

  68. 68

    Chen, V.B. et al. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D Biol. Crystallogr. 66, 12–21 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  69. 69

    Amunts, A. et al. Structure of the yeast mitochondrial large ribosomal subunit. Science 343, 1485–1489 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  70. 70

    Rappsilber, J., Mann, M. & Ishihama, Y. Protocol for micro-purification, enrichment, pre-fractionation and storage of peptides for proteomics using StageTips. Nat. Protoc. 2, 1896–1906 (2007).

    CAS  Google Scholar 

  71. 71

    Chen, Z.A. et al. Architecture of the RNA polymerase II-TFIIF complex revealed by cross-linking and mass spectrometry. EMBO J. 29, 717–726 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  72. 72

    Cox, J. & Mann, M. MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nat. Biotechnol. 26, 1367–1372 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

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Cryo-EM data were collected on a FEI Titan Krios at HHMI Janelia Farm. We also collected a cryo-EM data set on an FEI Technai F20 equipped with a K2 detector at NRAMM at the Scripps Research Institute, which is supported by NIH grant P41 GM103310. We thank Z. Yu, C. Hong, and R. Huang at HHMI, and C. Porter and B. Carragher at Scripps for help with data collection. H.L. dedicates this work to the loving memory of his son Paul J. Li. This work was funded by the US National Institutes of Health (grant GM111742 to H.L., and grant GM45436 to B.S.), the Biotechnology and Biological Sciences Research Council UK (grant P56061 to C. Speck), and the Wellcome Trust (Investigator Award P56628 to C. Speck, Senior Research Fellowship 103139 to J.R., Centre core grant 092076 to J.R., and instrument grant 108504 to J.R.).

Author information




Z.Y., A.R., L.B., J.S., J.R., Z.A.C., B.S., C. Speck, and H.L. designed experiments. Z.Y., A.R., L.B., S.N., C. Spanos, M.B., and J.S. performed experiments. Z.Y., A.R., L.B., J.S., Z.A.C., J.R., B.S., C. Speck, and H.L. analyzed the data. L.B., B.S., C. Speck, and H.L. wrote the manuscript with input from all other authors.

Corresponding authors

Correspondence to Juri Rappsilber or Bruce Stillman or Christian Speck or Huilin Li.

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Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Cryo-EM image processing and 3D-reconstruction procedure.

Motion-corrected raw particle images were sorted first by 2D classification. After removing raw particles that did not produce “good” averages, about 600,000 particles remained. These particles were further sorted into six 3D classes. Finally, particles that produced the best two 3D classes were combined, yielding ~300,000 particles for final 3D refinement. The final 3D map had an estimated resolution of 3.9 Å.

Supplementary Figure 2 Image resolution, particle Euler angle distribution, Fourier shell correlation, and local resolution map.

(a) Thon rings in the power spectra of a typical drift-corrected electron micrograph reached to 3.2 Å resolution. The upper left quadrant is a calculated version using the same CTF parameters as found in the experimental image. (b) The particle orientation covers all angular space. (c) Gold-standard Fourier correlation of two independent half maps. (d) Fourier shell correlations of the atomic model with the full 3D map (black), and the correlations of the 0.1-Å randomized and refined model against half map 1 (red), and with half map 2 (green), respectively. The 3.9 Å atomic model was randomly displaced by 0.1 Å. The noise-added model was refined by one round of coordinate and one round of b-factor refinement against half map 1. The refined coordinates were used to calculate FSC with half map 1, half map 2 and the full map respectively. The similarity between these curves indicates the atomic model is not over refined. (e) Local resolution map of the cryo-EM structure of OCCM complex in front side view (left), back side view (middle), and top ORC-Cdc6 view (right).

Supplementary Figure 3 Fitting of the atomic model with the cryo-EM density.

(a) Overall fitting of the OCCM model with the 3D map. (b) Fitting of four selected α-helices in ORC region. (c) Fitting of seven selected α-helices in the Cdt1-Mcm2-7 region.

Supplementary Figure 4 Cross-linking data are consistent with the cryo-EM structure of OCCM.

(a) Titration for cross-linking mass spectrometry. After a titration using a wide range of BS3 proportions, it was estimated that the best results were obtained between 1:5400 and 1:16000 protein-crosslinker ratios compared with the OCCM crosslinked with 2% glutaraldehyde. (b) Ten microliters of the final crosslinking reaction were run on a SDS PAGE before the cross-linking mass spectrometry analysis to confirm the homogeneity of the sample. (c) Atomic model of S. cerevisiae OCCM complex shown in surface view. The regions of Orc6 (grey), Mcm5 (yellow) and Orc2 (brown) that were not resolved in the atomic model, but covered with the cross-linking data are displayed as flat semi-transparent 2D surfaces delimited by a dashed line. The connector between the main body of Mcm3 and the Winged Helix Domain of the protein is presented in the same style. (d) A sketch of the OCCM structure in the same view as in (c). (e) Cross-links between ORC-Cdc6 and Cdt1-Mcm2-7 are shown as dashed green lines. (f) Histogram of alpha-carbon distances of observed cross-links as measured in the atomic model of the OCCM. Around 86% of the observed cross-links are within the allowed distance (less than 30 Å) while the rest can be explained due to the presence of flexible regions or large conformational changes in the complex.

Supplementary Figure 5 The dsDNA is loaded to the central channel of the Mcm2–7 hexamer.

(a) Experimental density map of dsDNA is colored in brown. (b) Resolution map of the dsDNA density isolated from OCCM complex. (c) Atomic model of dsDNA is superimposed onto the DNA density map. The front density of OCCM is removed to shown the DNA density in the middle.

Supplementary Figure 6 Nucleotide densities found at the Mcm4-Mcm7, Mcm6-Mcm4, Mcm7-Mcm3, Mcm2-Mcm6, Orc1-Orc4, Orc4-Orc5, Orc5-Orc3, and Cdc6-Orc1 interfaces.

Protein structures are shown in cartoon, ATPγS in sticks, and electron densities of the nucleotides are shown in gray meshes. The nucleotide density at the Orc4-Orc5 interface was the weakest among the eight sites. The Orc5-Orc3 interface was the smallest among the five Orc1-5 subunits, such that the complex could be divided into two sub-complexes of Orc1-Orc4-Orc5 and Orc3-Orc2. However, there was contact between Orc5 and Orc3 at the nucleotide-binding region such that the ATPγS density at the interface was clear.

Supplementary Figure 7 The position of the WHDs of individual Mcm proteins and their interaction with the ORC–Cdc6 loader ring.

(a) Six Mcm protein structures. Mcm2 has no WHD, and Mcm5 WHD was flexible in OCCM. (b) The positions of the two resolved WHDs in CMG helicase (Yuan, Z. et al. Nat Struct Mol Biol. 23, 217-224). No WHD was modeled in the reported DH structure (Li, N. et al. Nature, 524, 186-191). (c-f) Interaction of WHDs of Mcm3 (c), Mcm4 (d), Mcm6 (e) and Mcm7 (f) with ORC-Cdc6. Each of the four Mcm WHDs binds to one WHD of a loader ring subunit and an AAA-lid domain of its neighbor subunit simultaneously, but their individual binding mode is distinct.

Supplementary Figure 8 The closed Mcm2-Mcm5 gate at the C-tier AAA+ region, and analysis of the interaction between Cdt1 and Mcm2–7 subunits.

(a) Although the N-tier domains of Mcm2 and Mcm5 are separated by a gap, one α-helix in the Mcm5 AAA-RecA domain extends to and binds the AAA-RecA domain of Mcm2, indicating that the gate is closed at the C-tier region of Mcm2-7. (b) Cdt1 interacts with the Mcm2-7 complex as well as Mcm2, Mcm6 and Mcm7 subunits separately. Extracts from baculovirus infected Hi-Five cells containing Strep-Strep-SUMO tagged Cdt1 alone or with the Mcm2-7 complex or individual MCM subunits were precipitated with Strep-Tactin beads, washed, separated by gel electrophoresis and analyzed by silver-staining.

Supplementary Figure 9 Protein-DNA interaction and sequence alignment of Mcm2–7 at the DNA-binding regions.

(a) Detailed interaction between the protein subunits and the modeled 39-bp DNA. Left panel shows the first 20-bp DNA located in the top ORC-Cdc6 region. ORC-Cdc6 interacts with both strands. Right panel shows the remaining 19-bp DNA. The PS1 and/or H2I loops of Mcm2, Mcm4, Mcm6, and Mcm7 extensively interact with the last seven bases of the right strand. (b) Multi-sequence alignment of Mcm2-7. Regions underscored by green lines are the DNA-binding H2I and PS1 hairpins. Amino acids in blue directly contact DNA. The DNA-binding KA motif in the PS1 hairpin is highly conserved. The Mcm2, 4, 6, and 7 are grouped together to show the clustering of the DNA binding sites.

Supplementary Figure 10 Different DNA-binding modes between yeast and archaeal initiator proteins, and the surface charge of ORC–Cdc6.

(a) Comparison of the DNA binding model between the yeast Orc4 and archaeal homologue Orc1/Cdc6 PDB ID 2V1U). (b) Comparison of the DNA binding model of the yeast Cdc6 with the archaeal homologue Orc1/Cdc6. (c) Surface charge of the ORC-Cdc6 in top view that is distal to Cdt1-Mcm2-7, in bottom view that is proximal to the C-tier ring of Mcm2-7, in front side, and in back side view. The rounded rectangles mark several positive patches.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–10 (PDF 29248 kb)

Supplementary Data Set 1

Intra-molecular crosslinks of the OCCM complex detected by CLMS (CSV 23 kb)

Supplementary Data Set 2

Inter-molecular crosslinks of the OCCM complex detected by CLMS (CSV 6 kb)

Supplementary Video 1

Overall structure of the OCCM in complex with a 39-bp double-stranded DNA. (MOV 23897 kb)

Supplementary Video 2

Structural morph between the Drosophila apo-ORC and the S. cerevisiae ORC-Cdc6 in complex with DNA. The Orc3-Orc4-Orc5 region is similar in the two structures. The Orc1 AAA+ domain and the Orc2 WHD of the auto-inhibited DmORC need to move and flip by ~180° in order to match their respective yeast counterparts. The movements create a gap between Orc1 and Orc2 for DNA passage as well as for Cdc6 insertion. (MOV 3495 kb)

Supplementary Video 3

Structural morph of Mcm2-7 hexamer in S. cerevisiae OCCM-DNA complex into the structure in the S. cerevisiae Mcm2-7 double hexamer.First, the Cdt1 is removed to avoid steric clashes. The Mcm2-7 NTD ring needs to rotate by ~25° relative to the CTD ring in order to match the Mcm ring in the double hexamer. The CTDs of Mcm2 and Mcm5 need to rotate by ~5° and ~15°, respectively, to form the closed interface found in the double hexamer. After morphing to the double-hexamer configuration, the Mcm2-7 hexamer has no clash with the ORC-Cdc6 ring. This observation may explain why ORC-Cdc6 still binds the two head-to-head Mcm2-7 hexamers in the OCMM structure, an intermediate preceding the formation of the final loading product, the double hexamer (Sun, J. et al. Genes Dev. 28, 2291-2303 (2014)). (MOV 19808 kb)

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Yuan, Z., Riera, A., Bai, L. et al. Structural basis of Mcm2–7 replicative helicase loading by ORC–Cdc6 and Cdt1. Nat Struct Mol Biol 24, 316–324 (2017).

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