Abstract

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.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Accessions

Primary accessions

Electron Microscopy Data Bank

Protein Data Bank

References

  1. 1.

    & DNA replication in eukaryotic cells. Annu. Rev. Biochem. 71, 333–374 (2002).

  2. 2.

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

  3. 3.

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

  4. 4.

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

  5. 5.

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

  6. 6.

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

  7. 7.

    , & Crystal structure of the eukaryotic origin recognition complex. Nature 519, 321–326 (2015).

  8. 8.

    & Chromosome duplication in Saccharomyces cerevisiae. Genetics 203, 1027–1067 (2016).

  9. 9.

    , , & ATPase-dependent cooperative binding of ORC and Cdc6 to origin DNA. Nat. Struct. Mol. Biol. 12, 965–971 (2005).

  10. 10.

    & 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).

  11. 11.

    , , , & An essential role for the Cdc6 protein in forming the pre-replicative complexes of budding yeast. Nature 379, 180–182 (1996).

  12. 12.

    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).

  13. 13.

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

  14. 14.

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

  15. 15.

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

  16. 16.

    , & 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).

  17. 17.

    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).

  18. 18.

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

  19. 19.

    , , , & Uncoupling of sister replisomes during eukaryotic DNA replication. Mol. Cell 40, 834–840 (2010).

  20. 20.

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

  21. 21.

    , , , & The human GINS complex associates with Cdc45 and MCM and is essential for DNA replication. Nucleic Acids Res. 37, 2087–2095 (2009).

  22. 22.

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

  23. 23.

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

  24. 24.

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

  25. 25.

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

  26. 26.

    , & Phosphopeptide binding by Sld3 links Dbf4-dependent kinase to MCM replicative helicase activation. EMBO J. 35, 961–973 (2016).

  27. 27.

    , & Principles and concepts of DNA replication in bacteria, archaea, and eukarya. Cold Spring Harb. Perspect. Biol. 5, a010108 (2013).

  28. 28.

    , , , & Regulated eukaryotic DNA replication origin firing with purified proteins. Nature 519, 431–435 (2015).

  29. 29.

    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).

  30. 30.

    , , , & Single-molecule studies of origin licensing reveal mechanisms ensuring bidirectional helicase loading. Cell 161, 513–525 (2015).

  31. 31.

    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).

  32. 32.

    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).

  33. 33.

    , & Multiple functions for Mcm2-7 ATPase motifs during replication initiation. Mol. Cell 55, 655–665 (2014).

  34. 34.

    , , , & Origin licensing requires ATP binding and hydrolysis by the MCM replicative helicase. Mol. Cell 55, 666–677 (2014).

  35. 35.

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

  36. 36.

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

  37. 37.

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

  38. 38.

    , & 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).

  39. 39.

    , , & Structural basis of DNA replication origin recognition by an ORC protein. Science 317, 1213–1216 (2007).

  40. 40.

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

  41. 41.

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

  42. 42.

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

  43. 43.

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

  44. 44.

    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).

  45. 45.

    & On helicases and other motor proteins. Curr. Opin. Struct. Biol. 18, 243–257 (2008).

  46. 46.

    , , & AAA+: a class of chaperone-like ATPases associated with the assembly, operation, and disassembly of protein complexes. Genome Res. 9, 27–43 (1999).

  47. 47.

    , , & ATP hydrolysis by ORC catalyzes reiterative Mcm2-7 assembly at a defined origin of replication. Mol. Cell 16, 967–978 (2004).

  48. 48.

    , , & ATPase-dependent quality control of DNA replication origin licensing. Nature 495, 339–343 (2013).

  49. 49.

    & Clamp loaders and replication initiation. Curr. Opin. Struct. Biol. 16, 35–41 (2006).

  50. 50.

    , , & How a DNA polymerase clamp loader opens a sliding clamp. Science 334, 1675–1680 (2011).

  51. 51.

    , , & Replication origin recognition and deformation by a heterodimeric archaeal Orc1 complex. Science 317, 1210–1213 (2007).

  52. 52.

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

  53. 53.

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

  54. 54.

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

  55. 55.

    , & Mechanism of archaeal MCM helicase recruitment to DNA replication origins. Mol. Cell 61, 287–296 (2016).

  56. 56.

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

  57. 57.

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

  58. 58.

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

  59. 59.

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

  60. 60.

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

  61. 61.

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

  62. 62.

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

  63. 63.

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

  64. 64.

    , , , & MAGE-RING protein complexes comprise a family of E3 ubiquitin ligases. Mol. Cell 39, 963–974 (2010).

  65. 65.

    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).

  66. 66.

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

  67. 67.

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

  68. 68.

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

  69. 69.

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

  70. 70.

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

  71. 71.

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

  72. 72.

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

Download references

Acknowledgements

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

Author notes

    • Zuanning Yuan
    • , Alberto Riera
    •  & Lin Bai

    These authors contributed equally to this work.

Affiliations

  1. Cryo-EM Structural Biology Laboratory, Van Andel Research Institute, Grand Rapids, Michigan, USA.

    • Zuanning Yuan
    • , Lin Bai
    • , Jingchuan Sun
    •  & Huilin Li
  2. MRC London Institute of Medical Sciences (LMS), London, UK.

    • Alberto Riera
    • , Marta Barbon
    •  & Christian Speck
  3. DNA Replication Group, Institute of Clinical Sciences (ICS), Faculty of Medicine, Imperial College London, London, UK.

    • Alberto Riera
    • , Marta Barbon
    •  & Christian Speck
  4. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, USA.

    • Saikat Nandi
    •  & Bruce Stillman
  5. Wellcome Trust Centre for Cell Biology, University of Edinburgh, Edinburgh, UK.

    • Christos Spanos
    • , Zhuo Angel Chen
    •  & Juri Rappsilber
  6. Chair of Bioanalytics, Institute of Biotechnology, Technische Universität Berlin, Berlin, Germany.

    • Juri Rappsilber

Authors

  1. Search for Zuanning Yuan in:

  2. Search for Alberto Riera in:

  3. Search for Lin Bai in:

  4. Search for Jingchuan Sun in:

  5. Search for Saikat Nandi in:

  6. Search for Christos Spanos in:

  7. Search for Zhuo Angel Chen in:

  8. Search for Marta Barbon in:

  9. Search for Juri Rappsilber in:

  10. Search for Bruce Stillman in:

  11. Search for Christian Speck in:

  12. Search for Huilin Li in:

Contributions

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.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

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

Integrated supplementary information

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–10

CSV files

  1. 1.

    Supplementary Data Set 1

    Intra-molecular crosslinks of the OCCM complex detected by CLMS

  2. 2.

    Supplementary Data Set 2

    Inter-molecular crosslinks of the OCCM complex detected by CLMS

Videos

  1. 1.

    Supplementary Video 1

    Overall structure of the OCCM in complex with a 39-bp double-stranded DNA.

  2. 2.

    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.

  3. 3.

    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)).

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/nsmb.3372