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

Abstract

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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References

  1. 1

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

    CAS  Article  Google Scholar 

  2. 2

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

    CAS  Article  Google Scholar 

  3. 3

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

    CAS  Article  Google Scholar 

  4. 4

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

    CAS  Article  Google Scholar 

  5. 5

    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  Article  Google Scholar 

  6. 6

    Clarey, M.G. et al. Nucleotide-dependent conformational changes in the DnaA-like core of the origin recognition complex. Nat. Struct. Mol. Biol. 13, 684–690 (2006).

    CAS  Article  Google Scholar 

  7. 7

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

    CAS  Article  Google Scholar 

  8. 8

    Santocanale, C. & Diffley, J.F. ORC- and Cdc6-dependent complexes at active and inactive chromosomal replication origins in Saccharomyces cerevisiae. EMBO J. 15, 6671–6679 (1996).

    CAS  Article  Google Scholar 

  9. 9

    Donovan, S., Harwood, J., Drury, L.S. & Diffley, J.F. Cdc6p-dependent loading of Mcm proteins onto pre-replicative chromatin in budding yeast. Proc. Natl. Acad. Sci. USA 94, 5611–5616 (1997).

    CAS  Article  Google Scholar 

  10. 10

    Randell, J.C., Bowers, J.L., Rodriguez, H.K. & Bell, S.P. Sequential ATP hydrolysis by Cdc6 and ORC directs loading of the MCM2-7 helicase. Mol. Cell 21, 29–39 (2006).

    CAS  Article  Google Scholar 

  11. 11

    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  Article  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  Article  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  Article  Google Scholar 

  14. 14

    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  Article  Google Scholar 

  15. 15

    Ilves, I., Petojevic, T., Pesavento, J.J. & Botchan, M.R. Activation of the MCM2-7 helicase by association with Cdc45 and GINS proteins. Mol. Cell 37, 247–258 (2010).

    CAS  Article  Google Scholar 

  16. 16

    Kang, Y.H., Galal, W.C., Farina, A., Tappin, I. & Hurwitz, J. Properties of the human Cdc45/MCM2-7/GINS helicase complex and its action with DNA polymerase epsilon in rolling circle DNA synthesis. Proc. Natl. Acad. Sci. USA 109, 6042–6047 (2012).

    CAS  Article  Google Scholar 

  17. 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).

    CAS  Article  Google Scholar 

  18. 18

    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  Article  Google Scholar 

  19. 19

    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  Article  Google Scholar 

  20. 20

    Dueber, E.L., 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  Article  Google Scholar 

  21. 21

    Clarey, M.G., Botchan, M. & Nogales, E. Single particle EM studies of the Drosophila melanogaster origin recognition complex and evidence for DNA wrapping. J. Struct. Biol. 164, 241–249 (2008).

    CAS  Article  Google Scholar 

  22. 22

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

    CAS  Article  Google Scholar 

  23. 23

    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).

    CAS  Article  Google Scholar 

  24. 24

    Onesti, S. & MacNeill, S.A. Structure and evolutionary origins of the CMG complex. Chromosoma 122, 47–53 (2013).

    CAS  Article  Google Scholar 

  25. 25

    Forsburg, S.L. Eukaryotic MCM proteins: beyond replication initiation. Microbiol. Mol. Biol. Rev. 68, 109–131 (2004).

    CAS  Article  Google Scholar 

  26. 26

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

    CAS  Article  Google Scholar 

  27. 27

    Brewster, A.S. et al. Crystal structure of a near-full-length archaeal MCM: functional insights for an AAA+ hexameric helicase. Proc. Natl. Acad. Sci. USA 105, 20191–20196 (2008).

    CAS  Article  Google Scholar 

  28. 28

    Chesnokov, I.N., Chesnokova, O.N. & Botchan, M. A cytokinetic function of Drosophila ORC6 protein resides in a domain distinct from its replication activity. Proc. Natl. Acad. Sci. USA 100, 9150–9155 (2003).

    CAS  Article  Google Scholar 

  29. 29

    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).

    CAS  Article  Google Scholar 

  30. 30

    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  Article  Google Scholar 

  31. 31

    Pape, T. et al. Hexameric ring structure of the full-length archaeal MCM protein complex. EMBO Rep. 4, 1079–1083 (2003).

    CAS  Article  Google Scholar 

  32. 32

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

    CAS  Article  Google Scholar 

  33. 33

    Henderson, R. et al. Tilt-pair analysis of images from a range of different specimens in single-particle electron cryomicroscopy. J. Mol. Biol. 413, 1028–1046 (2011).

    CAS  Article  Google Scholar 

  34. 34

    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  Article  Google Scholar 

  35. 35

    Li, H., Chavan, M., Schindelin, H., Lennarz, W.J. & Li, H. Structure of the oligosaccharyl transferase complex at 12-Å resolution. Structure 16, 432–440 (2008).

    CAS  Article  Google Scholar 

  36. 36

    Lander, G.C. et al. Complete subunit architecture of the proteasome regulatory particle. Nature 482, 186–191 (2012).

    CAS  Article  Google Scholar 

  37. 37

    Lau, P.W., Potter, C.S., Carragher, B. & MacRae, I.J. DOLORS: versatile strategy for internal labeling and domain localization in electron microscopy. Structure 20, 1995–2002 (2012).

    CAS  Article  Google Scholar 

  38. 38

    Bochman, M.L., Bell, S.P. & Schwacha, A. Subunit organization of MCM2-7 and the unequal role of active sites in ATP hydrolysis and viability. Mol. Cell Biol. 28, 5865–5873 (2008).

    CAS  Article  Google Scholar 

  39. 39

    Pintilie, G.D., Zhang, J., Goddard, T.D., Chiu, W. & Gossard, D.C. Quantitative analysis of cryo-EM density map segmentation by watershed and scale-space filtering, and fitting of structures by alignment to regions. J. Struct. Biol. 170, 427–438 (2010).

    CAS  Article  Google Scholar 

  40. 40

    Fletcher, R.J. et al. The structure and function of MCM from archaeal M. Thermoautotrophicum. Nat. Struct. Biol. 10, 160–167 (2003).

    CAS  Article  Google Scholar 

  41. 41

    Liu, J. et al. Structure and function of Cdc6/Cdc18: implications for origin recognition and checkpoint control. Mol. Cell 6, 637–648 (2000).

    CAS  Article  Google Scholar 

  42. 42

    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).

    CAS  Article  Google Scholar 

  43. 43

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

    CAS  Article  Google Scholar 

  44. 44

    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  Google Scholar 

  45. 45

    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, 3162–3167 (2013).

    CAS  Article  Google Scholar 

  46. 46

    Kawasaki, Y., Kim, H.D., Kojima, A., Seki, T. & Sugino, A. Reconstitution of Saccharomyces cerevisiae prereplicative complex assembly in vitro. Genes Cells 11, 745–756 (2006).

    CAS  Article  Google Scholar 

  47. 47

    Ishimi, Y. A DNA helicase activity is associated with an MCM4, -6, and -7 protein complex. J. Biol. Chem. 272, 24508–24513 (1997).

    CAS  Article  Google Scholar 

  48. 48

    Bochman, M.L. & Schwacha, A. Differences in the single-stranded DNA binding activities of MCM2-7 and MCM467: MCM2 and MCM5 define a slow ATP-dependent step. J. Biol. Chem. 282, 33795–33804 (2007).

    CAS  Article  Google Scholar 

  49. 49

    Waga, S. & Stillman, B. The DNA replication fork in eukaryotic cells. Annu. Rev. Biochem. 67, 721–751 (1998).

    CAS  Article  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  Article  Google Scholar 

  51. 51

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

    CAS  Article  Google Scholar 

  52. 52

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

    CAS  Article  Google Scholar 

  53. 53

    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  Article  Google Scholar 

  54. 54

    Miyata, T. et al. Open clamp structure in the clamp-loading complex visualized by electron microscopic image analysis. Proc. Natl. Acad. Sci. USA 102, 13795–13800 (2005).

    CAS  Article  Google Scholar 

  55. 55

    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).

    CAS  Article  Google Scholar 

  56. 56

    Zou, L. & Stillman, B. Assembly of a complex containing Cdc45p, replication protein A, and Mcm2p at replication origins controlled by S-phase cyclin-dependent kinases and Cdc7p-Dbf4p kinase. Mol. Cell Biol. 20, 3086–3096 (2000).

    CAS  Article  Google Scholar 

  57. 57

    Klemm, R.D., Austin, R.J. & Bell, S.P. Coordinate binding of ATP and origin DNA regulates the ATPase activity of the origin recognition complex. Cell 88, 493–502 (1997).

    CAS  Article  Google Scholar 

  58. 58

    Baker, M.L., Zhang, J., Ludtke, S.J. & Chiu, W. Cryo-EM of macromolecular assemblies at near-atomic resolution. Nat. Protoc. 5, 1697–1708 (2010).

    CAS  Article  Google Scholar 

  59. 59

    Tang, G. et al. EMAN2: an extensible image processing suite for electron microscopy. J. Struct. Biol. 157, 38–46 (2007).

    CAS  Article  Google Scholar 

  60. 60

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

    CAS  Article  Google Scholar 

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Acknowledgements

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.

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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 or 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)

41594_2013_BFnsmb2629_MOESM27_ESM.mp4

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 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)

41594_2013_BFnsmb2629_MOESM28_ESM.mp4

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)

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). https://doi.org/10.1038/nsmb.2629

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