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Structure of the origin recognition complex bound to DNA replication origin

Nature volume 559, pages 217–222 (2018)Cite this article

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Abstract

The six-subunit origin recognition complex (ORC) binds to DNA to mark the site for the initiation of replication in eukaryotes. Here we report a 3 Å cryo-electron microscopy structure of the Saccharomyces cerevisiae ORC bound to a 72-base-pair origin DNA sequence that contains the ARS consensus sequence (ACS) and the B1 element. The ORC encircles DNA through extensive interactions with both phosphate backbone and bases, and bends DNA at the ACS and B1 sites. Specific recognition of thymine residues in the ACS is carried out by a conserved basic amino acid motif of Orc1 in the minor groove, and by a species-specific helical insertion motif of Orc4 in the major groove. Moreover, similar insertions into major and minor grooves are also embedded in the B1 site by basic patch motifs from Orc2 and Orc5, respectively, to contact bases and to bend DNA. This work pinpoints a conserved role of ORC in modulating DNA structure to facilitate origin selection and helicase loading in eukaryotes.

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Fig. 1: Overall structure of ORC bound to origin DNA.
Fig. 2: Extensive interactions between ORC and DNA around the ACS region.
Fig. 3: Base-specific recognition of ACS DNA by ORC.
Fig. 4: Interactions between ORC and DNA around the B1 element.
Fig. 5: Model of origin recognition and MCM loading by ORC.

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Acknowledgements

We thank the Electron Microscopy Laboratory of Peking University (cryo-EM platform) and the Tsinghua University Branch of the China National Center for Protein Sciences (Beijing) for the data collection of the ORC–DNA and apoORC samples, respectively. The computation was supported by High-performance Computing Platform of Peking University. This work was supported by the Ministry of Science and Technology of China (2016YFA0500700 to N.G.), the National Natural Science Foundation of China (NSFC) (31761163004, 31725007 and 31630087 to N.G.; 31700655 to N.L.), the Research Grants Council (RGC) of Hong Kong (GRF16138716 to B.K.T.; GRF16104115, GRF16143016 and GRF16104617 to Y.L.Z. and B.K.T.), NSFC/RGC Joint Research Scheme (N_HKUST614/17 to N.G., B.K.T., Y.L.Z. and N.L.), and the China Postdoctoral Science Foundation (2017M610013 to N.L.). N.L. is supported by Young Elite Scientists Sponsorship Program by CAST and a postdoctoral fellowship from the Peking-Tsinghua Centre for Life Sciences.

Reviewer information

Nature thanks C. Fox and the other anonymous reviewer(s) for their contribution to the peer review of this work.

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Author notes

  1. Yuanliang Zhai

    Present address: School of Biological Sciences, The University of Hong Kong, Hong Kong, China

  2. These authors contributed equally: Ningning Li, Wai Hei Lam, Yuanliang Zhai, Jiaxuan Cheng.

Authors and Affiliations

  1. State Key Laboratory of Membrane Biology, Peking-Tsinghua Center for Life Sciences, School of Life Sciences, Peking University, Beijing, China

    Ningning Li & Ning Gao

  2. Division of Life Science, The Hong Kong University of Science and Technology, Hong Kong, China

    Wai Hei Lam, Yuanliang Zhai, Yongqian Zhao & Bik-Kwoon Tye

  3. Institute of Advanced Study, The Hong Kong University of Science and Technology, Hong Kong, China

    Yuanliang Zhai & Yongqian Zhao

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

    Jiaxuan Cheng & Erchao Cheng

  5. Department of Molecular Biology & Genetics, Cornell University, Ithaca, NY, USA

    Bik-Kwoon Tye

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Contributions

Y.L.Z., N.G. and B.K.T. conceived the study; W.H.L., Y.L.Z. and Y.Q.Z. purified ORC; J.C., N.L., E.C., W.H.L. and Y.L.Z. prepared cryo grids; N.L. and J.C. collected data; N.L., J.C., Y.L.Z. and N.G. processed data; and N.L., W.H.L., Y.L.Z., N.G. and B.K.T. prepared the manuscript. N.L., W.H.L., Y.L.Z. and J.C. contributed equally to the study.

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Correspondence to Yuanliang Zhai, Ning Gao or Bik-Kwoon Tye.

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Extended data figures and tables

Extended Data Fig. 1 Sample preparation and image processing of the ORC–DNA complex.

a, SDS–PAGE analysis of the glycerol gradient fractions. ORC–DNA complexes (no fixation) were subjected to 10–30% glycerol gradient centrifugation. Fractions were collected and resolved on SDS–PAGE. Peak fractions (5–7) containing intact ORC complexes were processed for further electron microscopy analysis. Experiments were repeated multiple times (n > 10), and similar results were obtained. b, Negative-staining electron microscopy of the ORC–DNA complex. Samples from fraction 6 were subjected to negative staining. A severe dissociation of complexes was observed. c, Negative-staining electron microscopy of the ORC–DNA complex prepared with GraFix method using a gradient of glutaraldehyde (0–0.025%). d, A representative raw cryo-EM image of the ORC–DNA (72 bp) complex. e, 2D class averages of the ORC–DNA (72 bp) particles. f, Workflow of image processing of the ORC–DNA (72 bp) particles. The processing includes rounds of 2D classification, 3D classification, structural refinement and masked-based refinement procedures. g, FSC curves of the final density map of the ORC–DNA (72 bp) complex. h, The local resolution map of the final density map. i, Schematic domain organization of Orc1–Orc6 subunits. Regions that were built in the final atomic model were boxed in dashed grey lines. j–n, Local density of representative regions of the final cryo-EM density map, for Orc1-BP (j), Orc4-IH (k), the Orc1 ATP-binding pocket (l) and two other regions (m, n). For clarity, density of ATPγS is omitted in j to highlight the Walker A motif of Orc1.

Extended Data Fig. 2 Workflow of the image processing of the ORC–DNA (36 bp) and apoORC particles.

a, Image processing workflow of the ORC–DNA (36 bp) particles. Processing includes rounds of 2D classification, 3D classification, structural refinement and masked-based refinement procedures. b, Image processing workflow of the apoORC particles. c, FSC curves of the density maps of the ORC–DNA (36 bp) complex.

Extended Data Fig. 3 Organization of AAA+ and WHD domains around the origin DNA.

a, Organization of AAA+ domains of Orc1–Orc5 subunits around origin DNA. Cryo-EM maps of the AAA+ domains and DNA are shown in solid surface representation and colour-coded. The WHD of Orc2, which blocks the gap between the AAA+ domains of Orc1 and Orc2 is shown in cartoon representation. b, Same as in a, but for the WHDs of the Orc1–Orc5 subunits. c, Distribution of the HTH motifs of WHDs around the origin DNA. The WHDs of the Orc1–Orc5 subunits are shown in cartoon representation with the HTH motifs highlighted. d, Domain swapping between the AAA+ and WHD tiers. As shown, Orc2-WHD is in a different position from the rest. e, A flexible linker (residues 375–436) upstream of Orc1 AAA+ domains extends on the surface of Orc4 AAA+ domain (surface representation), with the further upstream basic patch sequences inserted into the minor groove of the ACS. The bound ATPγS is shown in stick model (orange). f, The very N-terminal extension (residues 15–50) of Orc3 wraps around the AAA+ domain of Orc2 (surface representation) and ends in the interface between the Orc2-WHD and Orc2-AAA+ domain. g, A very long N-terminal linker (NTD loop) upstream AAA+ domain of Orc2 extends on the surface of Orc3-WHD and TFIIB-B domain of Orc6. Note that the linker of Orc2 wrapping around Orc6 is traceable in the cryo-EM density map but the model could not be built at atomic level.

Extended Data Fig. 4 Configuration of the three ATPase centres in the ORC–DNA complex.

a, Zoomed-in view of the ATPase centre formed between Orc1 (O1) and Orc4 (O4). Orc1, Orc4 and ATPγS-Mg2+ are coloured blue, cyan and green, respectively. The Walker A and B motifs (WA and WB) of Orc1 and the arginine finger of Orc4 (R267) are highlighted in stick models. Inset, the stick model of ATPγS-Mg2+ superimposed with the cryo-EM density. b, Zoomed-in view of the ATPase centre formed between Orc4 and Orc5 (O5). Orc4, Orc5 and ATPγS-Mg2+ are coloured cyan, dark green and green, respectively. The Walker A and B motifs (WA and WB) of Orc4 and the equivalent arginine finger of Orc5 (R178) are highlighted in stick models. K151 of Orc5 within 4 Å distance from the γ-phosphate is shown. Inset, the stick model of ATPγS-Mg2+ superimposed with the cryo-EM density. c, Zoomed-in view of the ATPase centre formed between Orc5 and Orc3 (O3). Orc5, Orc3 and ATPγS-Mg2+ are coloured dark green, orange and green, respectively. The Walker A and B motifs of Orc5 are highlighted in stick models. Inset, the stick model of ATPγS-Mg2+ superimposed with the cryo-EM density. d, Comparison between the ATPase centres of O1:O4 and O4:O5, highlighting the flip of the base moiety of the bound ATPγS within the O4:O5 centre. The flip is forced by the replacement of a conserved glycine by a bulky tyrosine residue (Y107) of the Walker A motif of Orc4. The Walker A motifs were used as reference in the alignment. e, Comparison between the ATPase centres of the yeast O4:O5 and human O4:O5 (PDB code 5UJ7)32, highlighting the flip of the base moiety of the bound ATPγS within the yeast O4:O5 centre. The Walker A motifs were used as reference in the alignment. f, Sequence alignment of the Walker-A motif of Orc4 from different species.

Extended Data Fig. 5 Orc6 interacts with Orc2, Orc3 and Orc5.

a, b, Overview of the interactions between Orc3, Orc2, Orc5 and Orc6. c, d, Zoomed-in views of the boxed regions in a and b to highlight their relatively hydrophobic interfaces. Selected hydrophobic residues at the interface are displayed in stick model. A short helix in the linker between Orc6-CTD and Orc6-TFIIB-B packs with two helices from Orc2-AAA+ and Orc3-WHD (c). A long N-terminal linker of Orc2 (upstream the AAA+ domain) wraps the TFIIB-B domain of Orc6. Note that the linker of Orc2 (Orc2-NTD loop) is traceable in the cryo-EM density map but the model could not be built at atomic level. e, Low-pass filtered map of the ORC–DNA complex, highlighting the interactions (indicated by the presence of extra density) between the linker sequence of Orc6 (between TFIIB domains A and B) and DNA. The N-terminal (N-ter) end of the model built for Orc6 in our map is S217. f, Comparison between the yeast Orc6–TFIIB-B and human ORC6–TFIIB-B. The structure of human ORC6 is from a crystallography study (PDB code 3M03)65. The overall structure of the yeast ORC6–TFIIB-B is quite similar to its human counterpart. g, Superimposition of the structure of TFIIB-DNA onto the ORC–DNA complex. The crystal structure of a human TFIIB-TBP-DNA (PDB code 1VOL)66 was aligned using ORC6–TFIIB-B as reference. As shown, ORC6–TFIIB-B has not established extensive interactions with DNA. It is possible that further conformational change in Orc6 is required to form extensive interactions with DNA as the TFIIB does, probably at a later stage of replication licensing.

Extended Data Fig. 6 Flexibility of the ORC complexes.

a–c, Comparison of states I, II and III of the ORC–DNA complex (36 bp). Density maps of the three states are displayed in surface representation and in the Orc1–Orc2 side-view. The model of Orc2-WHD is highlighted in red cartoon. As shown, Orc2-WHD occupies different positions in the three maps. In the map of state II, the density of Orc2-WHD is relatively weak and it takes a position similar to that of the OCCM structure30. In the map of state III, Orc2-WHD is in a similar position as in state I, but its density is highly fragmented. Together, these indicate a floppy nature of the Orc2-WHD. d, Superimposition of the models of states I and II. The atomic model of state II was derived by flexible fitting of the state I model into the density map of state II. The alignment was done using Orc2 and Orc3. Compared with state I, the opening of the gap in the structure of state II is narrower. For clarity, the WHDs of Orc2 in the two states are omitted. e, Superimposition of the models of states I and III. The atomic model of state III was derived by flexible fitting of the state I model into the density map of state III. The alignment was done using Orc2 and Orc3. Compared with state I, the opening of the gap in the structure of state III is slightly larger. For clarity, the WHDs of Orc2 in the two states are omitted. f, g, Comparison of the density maps of states I and IV from the ORC–DNA (72 bp) dataset. A major difference between the two maps is the bending angle of the DNA. The extent of DNA bending correlates with the stability of Orc6 and Orc3 (Insertion domain of the AAA+ module). h, Top (left) and bottom (right) views of the cryo-EM map of the apoORC complex with the atomic model superimposed, which was derived by flexible fitting of the ORC–DNA model into the density map. ORC subunits are colour-coded. The AAA+ domain of Orc1 and the WHD of Orc2 are highly flexible, resulting in a large opening between Orc1 and Orc2, as indicated by the reduced EM densities of the corresponding regions.

Extended Data Fig. 7 Structural comparison between the S. cerevisiae ORC–DNA and the Drosophila apoORC complexes.

a, b, Side-by-side comparison of the yeast ORC–DNA and the Drosophila apoORC (PDB code 4XGC)31 complexes. a, The yeast ORC–DNA structure is shown in cartoon representation, with Orc1-AAA+ and Orc2-WHD highlighted in marine and blue, respectively. b, The Drosophila apoORC structure is shown in cartoon representation, with Orc1-AAA+ and Orc2-WHD highlighted in magenta and red, respectively. c, Superimposition of the yeast ORC–DNA and the Drosophila apoORC structures using Orc3–Orc5 as reference. Note that the positions and orientations of Orc1-AAA+ and Orc2-WHD are markedly different in the two structures. d, Superimposition of Orc1 from the structures of the yeast ORC–DNA and the Drosophila apoORC complexes using Orc1-AAA+ as a reference. Note that the Orc2-WHDs in two structures are in totally different positions relative to Orc1-AAA+, highlighting the distinct interfaces between Orc1-AAA+ and Orc2-WHD in the two structures.

Extended Data Fig. 8 Multiple sequence alignment of Orc1-BP, Orc4-IH, Orc5-BP and Orc2-BP.

a–d, Multi-sequence alignment of Orc1 N-terminal basic patches (a), Orc4 insertion helixes (b), Orc5 WHD basic patches (c) and Orc2 N-terminal basic patches (d) from various species as indicated. e, Multiple basic patches found between the BAH and AAA+ domains of Orc1 from yeast to human. The criteria for basic patches are a stretch of 10 to 14 amino acids flanked by either lysine or arginine with at least three basic (K or R) residues in between and a pair of them are spaced 3–4 residues apart as found in Orc1 (R367 and K362). f, Sequence information of the Orc1 basic patches in d from various species are listed as indicated.

Extended Data Fig. 9 Structural comparison between the yeast ORC–DNA and OCCM complexes.

a, b, AAA+ (a) and side (b) views of the ORC–DNA complex. ORC subunits and DNA are shown in cartoon representation and colour-coded. c, d, AAA+ (c) and side (d) views of the ORC in the context of the OCCM complex. ORC subunits and DNA are shown in cartoon representation and colour-coded. The OCCM structure (PDB code 5UDB) is from previous cryo-EM work30. Compared with the OCCM structure, ORC subunits of Orc1 and Orc2 in the structure of ORC–DNA are more compact around the DNA. Cdc6 (grey) is included in the side view. e, Relative orientation of the origin DNA with Orc1 in the ORC–DNA complex. Orc1-BP is inserted into the minor groove of ACS DNA. f, Same as in e, but for the DNA and Orc1 in OCCM complex. The distance between the AAA+ domain and DNA is considerably larger, resulting in the loss of DNA contact. g–i, Superimposition of the ORC–DNA (72 bp) and OCCM (PDB code 5UDB)30 structures. For clarity, ORC subunits from the OCCM is not shown. The Mcm2–Mcm7 subunits from the OCCM are shown in grey. Only Mcm2 and Mcm5 are labelled and colour-coded as indicated.

Extended Data Table 1 Cryo-EM data collection, refinement and validation statistics
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Supplementary information

Supplementary Information

This file contains the Supplementary Discussion and Supplementary Table 1.

Reporting Summary

Video 1: Structure of the ORC-DNA complex.

Segmented density of each ORC subunit is shown in transparent surface representation with atomic model superimposed. Interactions between DNA and ORC subunits are highlighted in zoom-in views. Next, the organization of ORC subunits and the potential DNA entry between Orc1 and Orc2 are shown. Last, the minor-groove inserting motif of Orc1-BP is highlighted in close-up views.

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Li, N., Lam, W.H., Zhai, Y. et al. Structure of the origin recognition complex bound to DNA replication origin. Nature 559, 217–222 (2018). https://doi.org/10.1038/s41586-018-0293-x

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