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

Nature Structural & Molecular Biology volume 24, pages 316–324 (2017)Cite this article

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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.

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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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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.).

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Author notes

  1. Zuanning Yuan, Alberto Riera and Lin Bai: These authors contributed equally to this work.

Authors and 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

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  1. Zuanning Yuan
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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.

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

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

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