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Crystal structure of the eukaryotic origin recognition complex

Abstract

Initiation of cellular DNA replication is tightly controlled to sustain genomic integrity. In eukaryotes, the heterohexameric origin recognition complex (ORC) is essential for coordinating replication onset. Here we describe the crystal structure of Drosophila ORC at 3.5 Å resolution, showing that the 270 kilodalton initiator core complex comprises a two-layered notched ring in which a collar of winged-helix domains from the Orc1–5 subunits sits atop a layer of AAA+ (ATPases associated with a variety of cellular activities) folds. Although canonical inter-AAA+ domain interactions exist between four of the six ORC subunits, unanticipated features are also evident. These include highly interdigitated domain-swapping interactions between the winged-helix folds and AAA+ modules of neighbouring protomers, and a quasi-spiral arrangement of DNA binding elements that circumnavigate an approximately 20 Å wide channel in the centre of the complex. Comparative analyses indicate that ORC encircles DNA, using its winged-helix domain face to engage the mini-chromosome maintenance 2–7 (MCM2–7) complex during replicative helicase loading; however, an observed out-of-plane rotation of more than 90° for the Orc1 AAA+ domain disrupts interactions with catalytic amino acids in Orc4, narrowing and sealing off entry into the central channel. Prima facie, our data indicate that Drosophila ORC can switch between active and autoinhibited conformations, suggesting a novel means for cell cycle and/or developmental control of ORC functions.

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Figure 1: Structure of Drosophila ORC.
Figure 2: Eukaryotic ORC and archaeal Orc WH domains.
Figure 3: AAA+/AAA+ domain interactions in ORC.
Figure 4: An unanticipated but naturally occurring Orc1 conformation.
Figure 5: The central channel in ORC probably binds DNA.
Figure 6: Model for ORC activation and its functional consequences.

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Accession codes

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Protein Data Bank

Data deposits

Atomic coordinates and structure factors have been deposited in the RSCB Protein Data Bank under accession code 4XGC.

References

  1. Siddiqui, K., On, K. F. & Diffley, J. F. Regulating DNA replication in eukarya. Cold Spring Harb. Perspect. Biol. 5, a012930 (2013)

    PubMed  PubMed Central  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)

    PubMed  PubMed Central  Google Scholar 

  3. Bell, S. P. & Kaguni, J. M. Helicase loading at chromosomal origins of replication. Cold Spring Harb. Perspect. Biol. 5, a010124 (2013)

    PubMed  PubMed Central  Google Scholar 

  4. 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  Google Scholar 

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

    ADS  CAS  PubMed  Google Scholar 

  6. Diffley, J. F. & Cocker, J. H. Protein-DNA interactions at a yeast replication origin. Nature 357, 169–172 (1992)

    ADS  CAS  PubMed  Google Scholar 

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

    CAS  Google Scholar 

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

    CAS  Google Scholar 

  9. Iyer, L. M., Leipe, D. D., Koonin, E. V. & Aravind, L. Evolutionary history and higher order classification of AAA+ ATPases. J. Struct. Biol. 146, 11–31 (2004)

    CAS  PubMed  Google Scholar 

  10. 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)

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  11. Liu, S. et al. Structural analysis of human Orc6 protein reveals a homology with transcription factor TFIIB. Proc. Natl Acad. Sci. USA 108, 7373–7378 (2011)

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

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

    CAS  PubMed  PubMed Central  Google Scholar 

  13. Yardimci, H. & Walter, J. C. Prereplication-complex formation: a molecular double take? Nature Struct. Mol. Biol. 21, 20–25 (2014)

    CAS  Google Scholar 

  14. 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 

  15. 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)

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  16. 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)

    ADS  CAS  PubMed  Google Scholar 

  17. 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)

    ADS  CAS  PubMed  Google Scholar 

  18. Singleton, M. R. et al. Conformational changes induced by nucleotide binding in Cdc6/ORC from Aeropyrum pernix. J. Mol. Biol. 343, 547–557 (2004)

    CAS  PubMed  Google Scholar 

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

    CAS  PubMed  Google Scholar 

  20. Dueber, E. C. et al. Molecular determinants of origin discrimination by Orc1 initiators in archaea. Nucleic Acids Res. 39, 3621–3631 (2011)

    CAS  PubMed  PubMed Central  Google Scholar 

  21. Robinson, N. P. et al. Identification of two origins of replication in the single chromosome of the archaeon Sulfolobus solfataricus. Cell 116, 25–38 (2004)

    CAS  PubMed  Google Scholar 

  22. Erzberger, J. P., Mott, M. L. & Berger, J. M. Structural basis for ATP-dependent DnaA assembly and replication-origin remodeling. Nature Struct. Mol. Biol. 13, 676–683 (2006)

    CAS  Google Scholar 

  23. 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 

  24. Sun, J. et al. Cryo-EM structure of a helicase loading intermediate containing ORC–Cdc6–Cdt1–MCM2–7 bound to DNA. Nature Struct. Mol. Biol. 20, 944–951 (2013)

    CAS  Google Scholar 

  25. Ranjan, A. & Gossen, M. A structural role for ATP in the formation and stability of the human origin recognition complex. Proc. Natl Acad. Sci. USA 103, 4864–4869 (2006)

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  26. Siddiqui, K. & Stillman, B. ATP-dependent assembly of the human origin recognition complex. J. Biol. Chem. 282, 32370–32383 (2007)

    CAS  PubMed  Google Scholar 

  27. 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  PubMed  Google Scholar 

  28. Chesnokov, I., Remus, D. & Botchan, M. Functional analysis of mutant and wild-type Drosophila origin recognition complex. Proc. Natl Acad. Sci. USA 98, 11997–12002 (2001)

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  29. Giordano-Coltart, J., Ying, C. Y., Gautier, J. & Hurwitz, J. Studies of the properties of human origin recognition complex and its Walker A motif mutants. Proc. Natl Acad. Sci. USA 102, 69–74 (2005)

    ADS  CAS  PubMed  Google Scholar 

  30. Kong, D., Coleman, T. R. & DePamphilis, M. L. Xenopus origin recognition complex (ORC) initiates DNA replication preferentially at sequences targeted by Schizosaccharomyces pombe ORC. EMBO J. 22, 3441–3450 (2003)

    CAS  PubMed  PubMed Central  Google Scholar 

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

    ADS  CAS  PubMed  Google Scholar 

  32. Duderstadt, K. E., Chuang, K. & Berger, J. M. DNA stretching by bacterial initiators promotes replication origin opening. Nature 478, 209–213 (2011)

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  33. 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  Google Scholar 

  34. 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)

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  35. Simonetta, K. R. et al. The mechanism of ATP-dependent primer-template recognition by a clamp loader complex. Cell 137, 659–671 (2009)

    CAS  PubMed  PubMed Central  Google Scholar 

  36. Remus, D., Beall, E. L. & Botchan, M. R. DNA topology, not DNA sequence, is a critical determinant for Drosophila ORC-DNA binding. EMBO J. 23, 897–907 (2004)

    CAS  PubMed  PubMed Central  Google Scholar 

  37. Vashee, S. et al. Sequence-independent DNA binding and replication initiation by the human origin recognition complex. Genes Dev. 17, 1894–1908 (2003)

    CAS  PubMed  PubMed Central  Google Scholar 

  38. Houchens, C. R. et al. Multiple mechanisms contribute to Schizosaccharomyces pombe origin recognition complex-DNA interactions. J. Biol. Chem. 283, 30216–30224 (2008)

    CAS  PubMed  PubMed Central  Google Scholar 

  39. Gaczynska, M. et al. Atomic force microscopic analysis of the binding of the Schizosaccharomyces pombe origin recognition complex and the spOrc4 protein with origin DNA. Proc. Natl Acad. Sci. USA 101, 17952–17957 (2004)

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  40. Balasov, M., Huijbregts, R. P. & Chesnokov, I. Role of the Orc6 protein in origin recognition complex-dependent DNA binding and replication in Drosophila melanogaster. Mol. Cell. Biol. 27, 3143–3153 (2007)

    CAS  PubMed  PubMed Central  Google Scholar 

  41. Remus, D., Blanchette, M., Rio, D. C. & Botchan, M. R. CDK phosphorylation inhibits the DNA-binding and ATP-hydrolysis activities of the Drosophila origin recognition complex. J. Biol. Chem. 280, 39740–39751 (2005)

    CAS  PubMed  Google Scholar 

  42. DePamphilis, M. L. Cell cycle dependent regulation of the origin recognition complex. Cell Cycle 4, 70–79 (2005)

    CAS  PubMed  Google Scholar 

  43. Chuang, R. Y. & Kelly, T. J. The fission yeast homologue of Orc4p binds to replication origin DNA via multiple AT-hooks. Proc. Natl Acad. Sci. USA 96, 2656–2661 (1999)

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  44. Kuo, A. J. et al. The BAH domain of ORC1 links H4K20me2 to DNA replication licensing and Meier–Gorlin syndrome. Nature 484, 115–119 (2012)

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  45. Muller, P. et al. The conserved bromo-adjacent homology domain of yeast Orc1 functions in the selection of DNA replication origins within chromatin. Genes Dev. 24, 1418–1433 (2010)

    CAS  PubMed  PubMed Central  Google Scholar 

  46. Onishi, M. et al. Role of the conserved Sir3-BAH domain in nucleosome binding and silent chromatin assembly. Mol. Cell 28, 1015–1028 (2007)

    CAS  PubMed  Google Scholar 

  47. 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)

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  48. 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 

  49. Parks, T. D. et al. Release of proteins and peptides from fusion proteins using a recombinant plant virus proteinase. Anal. Biochem. 216, 413–417 (1994)

    CAS  PubMed  Google Scholar 

  50. Kabsch, W. Xds. Acta Crystallogr. D 66, 125–132 (2010)

    CAS  PubMed  PubMed Central  Google Scholar 

  51. Kabsch, W. Integration, scaling, space-group assignment and post-refinement. Acta Crystallogr. D 66, 133–144 (2010)

    CAS  PubMed  PubMed Central  Google Scholar 

  52. Winn, M. D. et al. Overview of the CCP4 suite and current developments. Acta Crystallogr. D 67, 235–242 (2011)

    CAS  PubMed  PubMed Central  Google Scholar 

  53. Sheldrick, G. M. Experimental phasing with SHELXC/D/E: combining chain tracing with density modification. Acta Crystallogr. D 66, 479–485 (2010)

    CAS  PubMed  PubMed Central  Google Scholar 

  54. McCoy, A. J. et al. Phaser crystallographic software. J. Appl. Cryst. 40, 658–674 (2007)

    CAS  Google Scholar 

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

    CAS  PubMed  PubMed Central  Google Scholar 

  56. Terwilliger, T. C. Maximum-likelihood density modification. Acta Crystallogr. D 56, 965–972 (2000)

    CAS  PubMed  PubMed Central  Google Scholar 

  57. Vonrhein, C., Blanc, E., Roversi, P. & Bricogne, G. Automated structure solution with autoSHARP. Methods Mol. Biol. 364, 215–230 (2007)

    CAS  PubMed  Google Scholar 

  58. Bricogne, G. et al. Generation, representation and flow of phase information in structure determination: recent developments in and around SHARP 2.0. Acta Crystallogr. D 59, 2023–2030 (2003)

    CAS  PubMed  Google Scholar 

  59. Vagin, A. & Teplyakov, A. Molecular replacement with MOLREP. Acta Crystallogr. D 66, 22–25 (2010)

    CAS  PubMed  Google Scholar 

  60. Goddard, T. D., Huang, C. C. & Ferrin, T. E. Visualizing density maps with UCSF Chimera. J. Struct. Biol. 157, 281–287 (2007)

    CAS  PubMed  Google Scholar 

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

    CAS  PubMed  Google Scholar 

  62. Kelley, L. A. & Sternberg, M. J. Protein structure prediction on the Web: a case study using the Phyre server. Nature Protocols 4, 363–371 (2009)

    CAS  PubMed  Google Scholar 

  63. Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D 66, 486–501 (2010)

    CAS  PubMed  PubMed Central  Google Scholar 

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

    CAS  PubMed  Google Scholar 

  65. Katoh, K., Kuma, K., Toh, H. & Miyata, T. MAFFT version 5: improvement in accuracy of multiple sequence alignment. Nucleic Acids Res. 33, 511–518 (2005)

    CAS  PubMed  PubMed Central  Google Scholar 

  66. Katoh, K. & Toh, H. Recent developments in the MAFFT multiple sequence alignment program. Brief. Bioinform. 9, 286–298 (2008)

    CAS  PubMed  Google Scholar 

  67. Ashkenazy, H. et al. ConSurf 2010: calculating evolutionary conservation in sequence and structure of proteins and nucleic acids. Nucleic Acids Res. 38, W529–W533 (2010)

    CAS  PubMed  PubMed Central  Google Scholar 

  68. Crooks, G. E., Hon, G., Chandonia, J. M. & Brenner, S. E. WebLogo: a sequence logo generator. Genome Res. 14, 1188–1190 (2004)

    CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We thank the beamline scientists at beamlines 8.3.1 of the Advanced Light Source (Berkeley Lawrence National Laboratory), 23-ID-B of the Advanced Photon Source (Argonne National Laboratory) and X25 of the National Synchrotron Light Source (Brookhaven National Laboratory) for technical support with X-ray diffraction data collection, and the Nogales laboratory (University of California, Berkeley) for EM support. We also thank A. Fisher and Y. Li for assistance with insect cell cultures. This work was supported by the National Institutes of Health (GM071747 to J.M.B. and CA R37-30490 to M.R.B.) and by a fellowship from the UC Berkeley Miller Institute for Basic Research in Science (to F.B.).

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Authors

Contributions

F.B. performed all biochemical and crystallization experiments, collected X-ray diffraction data and determined the structure with guidance from J.M.B. All authors interpreted and discussed results, and wrote the manuscript.

Corresponding authors

Correspondence to Michael R. Botchan or James M. Berger.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Deletion of variable N-terminal extensions in Orc1, Orc2 and Orc3 alters neither ORC stability nor overall ORC architecture.

a, Gel-filtration chromatography trace of the ORC core used for crystallography, with (b) SDS–PAGE of respective ORC peak fractions from a, indicates the formation of a stable hexameric complex. c, Full-length ORC and ORC containing N-terminal truncations display similar structural features in two-dimensional EM class averages. Both complexes were imaged by negative-stain EM in the presence of ATPγS. Note that although class averages from ORC with truncated Orc1–3 subunits contain full-length Orc6, Orc6 is not visible owing to its flexible nature14. Class averages for full-length ORC are derived from a data set used in ref. 14.

Extended Data Figure 2 Experimental electron density contoured at 1σ for different regions of the ORC structure.

The Orc1 WH domain is shown in a, the Orc3 insertion in b and the Orc4 AAA+ domain in c.

Extended Data Figure 3 Comparison of individual ORC subunits and archaeal Orc structures.

Structures of individual ORC subunits are compared to S. solfataricus Orc1-1 (PDB accession number 2QBY chain A16) and A. pernix Orc2 (PDB accession numbers 1W5S chain A (left) and 1W5T chain C (right)18). Different structural elements are coloured as indicated. The initiator-specific motif (ISM) of the AAA+ ATPase fold is shown in the inset. No electron density was observed for the region linking the AAA+ and WH domains of Orc5 (indicated by a dashed line). The very N-terminal region of Orc2, which could only be built as stretches of polyalanine, is not shown.

Extended Data Figure 4 The Orc3 domain insertion forms a conserved, hydrophobic binding platform for Orc6.

a, Surface representation of ORC. The Orc3 insertion, which extends from the Orc3 AAA+ lid subdomain and interacts with the C-terminal helix of Orc6, is boxed. b, Secondary structure representation of the boxed region shown in a. The Orc3 insertion forms a bi-lobed, α-helical fold, three helices of which create a binding site for Orc6. c, Surface conservation of the Orc3 insertion. Conserved Orc3 residues cluster in the region that interacts with the Orc3 lid and in the Orc6 binding pocket. The latter region contacts highly conserved residues in Orc6 (Y225 and W228). d, Close-up view of Orc3•Orc6 interactions, showing a primarily hydrophobic binding site in Orc3 for Orc6 residues (Y225, W228, M232, A236). Y225, which in Drosophila Orc6 is equivalent to an amino acid altered in a subset of patients with Meier–Gorlin syndrome, appears positioned within hydrogen-bonding distance of E354 in Orc3 (dashed line). Colours are as in b. eh, Biochemical validation of the binding register for Drosophila Orc6. e, Close-up of the Orc6•Orc3 interface. Orc6–Ala236 faces a hydrophobic surface formed by Orc3 residues and is also in close proximity to a natural cysteine in Orc3 (Cys372). To validate the register of the short C-terminal Orc6 helix and the Orc6•Orc3 interface, we mutated Orc6–Ala236 to either glutamate, which we hypothesized would impede binding to ORC1–5 because of clashes with hydrophobic residues in Orc3, or to cysteine, which we presumed would not affect Orc3 binding but would allow site-specific crosslinking to Orc3-Cys372. f, Orc6A236E has a reduced affinity for the ORC1–5 complex. The C-terminal domains (CTDs) of wild-type (WT) Orc6, Orc6A236E or the Meier–Gorlin syndrome equivalent Orc6Y225S were each N-terminally labelled with Alexa Fluor 488 and tested for ORC1–5 binding using fluorescence anisotropy. As shown previously14, the C-terminal domain of Orc6 binds ORC1–5 with low nanomolar affinity, whereas the Y225S mutation strongly reduces binding. As predicted on the basis of the structure of the Orc6•Orc3 interface, the A236E mutation also reduces the affinity of the Orc6-CTD for ORC1–5. Mean and standard deviations from three (for Orc6Y225S and Orc6A236E) or six (for wild-type Orc6) independent experiments are shown. g, Orc6A236C is able to bind to the ORC1–5 complex. Orc6-CTDWT or Orc6-CTDA236C were incubated with ORC1–5 (containing MBP-tagged Orc4) and subjected to pull-down experiments using amylose resin. Both Orc6-CTDWT and Orc6-CTDA236C co-purified with ORC1–5. The pull-down experiment was performed under non-reducing experimental conditions similar to the crosslinking experiment in h. Asterisks mark two likely proteolytic fragments of Orc3. h, The Orc6-CTDA236C mutant, but not the wild-type Orc6-CTD, specifically crosslinks to Orc3 within the ORC1–5 complex. Orc6-CTDWT or Orc6-CTDA236C, either alone or in the presence of ORC1–5, was incubated with a bifunctional maleimide crosslinker and the proteins subsequently analysed by SDS–PAGE. In reactions containing ORC1–5 and Orc6-CTDA236C, crosslinking gives rise to a novel band with higher molecular mass than Orc3; the appearance of this band correlates with a decrease in the amount of uncrosslinked Orc3 and Orc6-CTD, and does not appear with reactions containing ORC1–5 and wild-type Orc6-CTD, indicating that this species corresponds to an Orc3–Orc6 crosslink (a moderately strong higher molecular-mass band that appears in the absence of Orc6 probably corresponds to homotypic adducts between exposed cysteines in Orc3). These results are consistent with the structure, which places Orc6-Ala236 in close proximity to Orc3-Cys372. Note that ORC1–5 contained MBP-tagged Orc4 in g but that the tag was removed in h.

Extended Data Figure 5 ATP-binding site configuration at the Orc4•Orc5 and Orc5•Orc3 interfaces.

a, Inter-AAA+ interactions between Orc4 and Orc5 are similar to canonical AAA+ interactions between DnaA protomers (top panel, only Orc4 is used for superpositioning onto the left (light grey) AAA+ domain of an ATP-bound DnaA dimer, PDB accession number 2HCB22). Close-up views of the nucleotide-binding site are shown for Orc4 (bottom panel) and for DnaA for comparison (middle panel). The resemblance of the Orc4 nucleotide-binding pocket to the active site of functional AAA+ ATPases is somewhat surprising considering that mutations in the active site of Drosophila and human Orc4 have no reported effect on the ATPase activity of ORC as measured in vitro28,29, but may help explain why a Drosophila ORC mutant bearing a Walker A or B substitution in Orc4 exhibits modest DNA replication defects in extracts28. b, The putative arginine finger in Orc5 is well conserved across homologues. A sequence logo of a multiple sequence alignment of the region containing the putative arginine finger (marked with an arrow) in eukaryotic Orc5 protein sequences is shown. Amino-acid numberings correspond to the Drosophila Orc5 sequence. c, A potential Sensor II equivalent arginine (marked with an arrow) in the Orc4 Walker A motif is conserved in eukaryotic Orc4 homologues. A sequence logo of the Walker A motif from a multiple sequence alignment of eukaryotic Orc4 protein sequences is shown. Amino-acid positions are numbered as in Drosophila Orc4. d, Inter-AAA+ interactions between Orc5 and Orc3. The top panel shows a superposition derived from placing the AAA+ domain of Orc5 atop the AAA+ domain of the left (dark grey) protomer of an ATP-bound DnaA dimer; the bottom panel shows a close-up view of the nucleotide-binding site at the Orc5•Orc3 interface. Side chains of conserved residues known to be involved in nucleotide binding and hydrolysis in AAA+ ATPases are represented as sticks in both a and d. WA, Walker A; WB, Walker B; SI, Sensor I; SII, Sensor II; RF, arginine finger.

Extended Data Figure 6 The conformation of Orc1 arises from a reorientation between its AAA+ and WH domains, not from changes within the AAA+ ATPase domain itself.

a, Superpositioning of the WH domains of Orc1 and S. solfataricus Orc1-1 (PDB accession number 2QBY chain A16) reveals different conformations for both proteins, resulting from a large domain rotation of the Orc1 AAA+ domain around a pivot point in the linker preceding its WH domain. b, The Orc1 conformation is most similar to a state seen for A. pernix Orc2 (PDB accession number 1W5T chain C18). The WH domains of both proteins were superposed as in a. ce, Superposing the AAA+ base subdomains of Orc1 and S. solfataricus Orc1-1 (c, PDB accession number 2QBY chain A16), A. pernix Orc2 (d, PDB accession number 1W5T chain C18) and Orc3, Orc4 or Orc5 (e) shows that the typical AAA+ configuration between the base and lid subdomains are maintained in Orc1. Only a slight opening of the nucleotide-binding cleft is observed in Orc1, which is probably caused by the absence of bound nucleotide. f, g, The most C-terminal α-helix of the Orc1 WH domain mediates interactions with the Orc1 lid subdomain. An overview of the interaction is shown in f, with a close-up view of contacts between a conserved tyrosine (Tyr915) in the C-terminal Orc1 helix and a hydrophobic pocket of the Orc1 lid depicted in g. h, The tyrosine in the C-terminal helix of Orc1 is well conserved across metazoan but not fungal Orc1 homologues. Alignments are shown as sequence logos. The numbering of amino acids is based on Drosophila Orc1, and the tyrosine is marked by an arrow.

Extended Data Figure 7 Nucleotide binding by Orc1, Orc4 and Orc5.

For ac, molecular replacement with the apo-ORC model was used to phase diffraction data collected from an ORC–ATPγS co-crystal. Positive Fo − Fc difference density contoured at different sigma levels reveals clear features for nucleotide binding to the AAA+ domains of (a) Orc1, (b) Orc4 and (c) Orc5. ATPγS is docked into the difference density for reference; owing to the moderate (4.0 Å) resolution of the data, this structure was not refined. d, Modelling of canonical AAA+ interactions between Orc1 and Orc4, generated using the Orc4•Orc5 interaction as a reference. Upper panel: structural overview of modelled AAA+ domain positioning between Orc1 and Orc4. Lower panel: close-up of the modelled Orc1•Orc4 ATPase site. Side chains (taken from their place in the apo-ORC model as a reference) are shown for conserved catalytically important residues.

Extended Data Figure 8 Comparison of crystallographic and EM models.

Docking of the observed and remodelled ORC structures into the cryo-EM density of S. cerevisiae and Drosophila ORC indicates that the ATPase domain of Orc1 is repositioned into a canonical AAA+/AAA+ interaction with Orc4 when Cdc6 is present, and supports a model where DNA passes through the central channel in ORC. a, The three-dimensional EM volume for S. cerevisiae ORC (as present in a complex with Cdc6, Cdt1 and MCM2–7 and assembled in the presence of DNA, EMD-5625 (ref. 24)) contains Orc1 in the activated conformation. ORC with Orc1 in the autoinhibited conformation (left panel, as observed in the crystal structure) and remodelled conformation (right panel, remodelled) were docked into the ORC•Cdc6•Cdt1•Mcm2-7 cryo-EM map (only the density for ORC•Cdc6 is shown). The ORC•Cdc6 EM density readily accommodates Orc1 in the activated conformation, but not in its autoinhibited state. The EM density corresponding to Cdc6 is indicated in the right panel. b, DNA passes through the central ORC channel in the DNA•ORC•Cdc6 complex. ORC (with Orc1 in the remodelled conformation) was first docked into the cryo-EM map derived from a DNA•ORC•Cdc6 complex (EMD-5381 (ref. 23)). The AAA+ domain and its associated DNA from either S. solfataricus Orc1-1 or Orc1-3 (PDB accession number 2QBY16) were then superposed using the AAA+ domain of Orc4 as a guide. Both dockings indicate that a region of density previously assigned to the Orc6 subunit23 actually corresponds to the DNA duplex. Although superpositioning of the AAA+ domain of S. solfataricus Orc1-3 onto Orc4 better positions duplex DNA in the observed EM density (than does the comparable exercise using the Orc1-1•DNA complex), the curvature of the DNA (as present in the Orc1-3•DNA co-crystal structure) results in a greater number of clashes between DNA and ORC subunits. Nevertheless, both docking scenarios are consistent with a DNA binding mode of ORC where DNA runs through the central channel. Note that the handedness of the EM map (EMD-5381 (ref. 23)) has been corrected in this figure because it has been reported that the original handedness was inverted24. For clarity, the WH domain of Orc2 is omitted from the remodelled ORC structure in b. c, The autoinhibited ORC conformation observed in the crystal is, unlike the remodelled Orc1 configuration, similar to the ORC conformation observed in Drosophila ORC EM reconstructions. Docking of the ORC crystal structure (top panels) or the remodelled activated ORC structure (bottom panels) into a prior three-dimensional EM reconstruction of Drosophila ORC (EMD-2479)14 reveals excellent agreement between EM and crystal structures, but not between EM and modelled activated ORC structures. The poor fit of the remodelled Orc1 conformation into the EM density suggests that the EM structure represents the autoinhibited state of ORC as seen in the crystal, indicating it is the predominant state in solution. See also Supplementary Video 2.

Extended Data Figure 9 Docking of the ORC structure into the cryo-EM structure of an S. cerevisiae replication initiation intermediate indicates that ORC recruits the MCM2–7 complex by binding to the ORC WH domains.

A prior model for ORC•MCM2–7 engagement24, proposed from an ORC•Cdc6•Cdt1•MCM2–7 cryo-EM structure generated in the presence of DNA (shown in a, EMD-5625 (ref. 24)), used the crystal structure of replication factor C (RFC) bound to the sliding clamp PCNA (shown in b, PDB accession number 1SXJ31) to suggest that the AAA+ domains of ORC engage the MCM2–7•Cdt1 complex. However, using the handedness of the EM volume as reported24, this organization of ORC subunits leads to an inverted ATPase site assembly, requiring that the Orc4 arginine finger (which is known to stimulate Orc1 ATP hydrolysis33) points towards the Orc5 nucleotide-binding site rather than the appropriate Orc1 active site. Schematics for the ATP site assemblies of ORC and RFC derived from these structures are shown in the lower panels in a and b. The location of the WH domain collar of ORC and the C-terminal collar of RFC is indicated by a grey circle. c, Docking of the ORC crystal structure (with Orc1 in its remodelled or ‘activated’ conformation) into the cryo-EM map shown in a reveals that the WH domains of ORC face an MCM2–7 complex. This switched polarity of WH domains and AAA+ domains in the EM map corrects the ATPase site assembly and is schematized in the right panel.

Extended Data Table 1 Summary of data collection, phasing and refinement statistics

Supplementary information

Supplementary Information

DESCRIPTION (PDF 138 kb)

Structure of Drosophila ORC

In the video, ORC starts off in the autoinhibited state as observed in crystals. ORC is first shown in the cartoon representation with individual subunits colored differentially (Orc1 – orange, Orc2 – green, Orc3 – blue, Orc4 – purple, Orc5 – gold, Orc6 – deepsalmon). Subsequently, the molecular surface is shown with the WH domains in light tints of their respective AAA+ domain color to illustrate domain swapping, which is best visualized in the side views with the Orc1 AAA+ and Orc2 WH domains removed. Remaining in the side view, the Orc1 AAA+ domain is shown next, which then morphs between autoinhibited (as observed in the crystal) and prospective remodeled (“activated”) conformations. The remodeled structure is then shown in cartoon format, with the ISMs and β-hairpin wings of ORC subunits rendered as surface in red and cyan, respectively. A close-up view of the central channel is next provided, illustrating the shallow spiral formed by the ISMs and β-hairpin wings. Finally, a 360° view of ORC in the remodeled conformation is shown in cartoon representation. (MP4 24801 kb)

Comparison of Drosophila ORC crystal and EM structures

The ORC crystal structure (Orc1 – orange, Orc2 – green, Orc3 – blue, Orc4 – purple, Orc5 – gold, Orc6 – deepsalmon) is docked into the ATPγS-bound Drosophila ORC EM volume (gray surface, EMD-247914). In the second part of the video, Orc1 is morphed into a remodeled (“activated”) conformation. The close agreement between the EM map and the ORC crystal structure, but not the remodeled ORC conformation, suggests that the autoinhibited state of ORC as observed in crystals corresponds to the predominant species in solution. (MP4 5252 kb)

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Bleichert, F., Botchan, M. & Berger, J. Crystal structure of the eukaryotic origin recognition complex. Nature 519, 321–326 (2015). https://doi.org/10.1038/nature14239

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