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. 2013 Aug;20(8):944-51.
doi: 10.1038/nsmb.2629. Epub 2013 Jul 14.

Cryo-EM Structure of a Helicase Loading Intermediate Containing ORC-Cdc6-Cdt1-MCM2-7 Bound to DNA

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Cryo-EM Structure of a Helicase Loading Intermediate Containing ORC-Cdc6-Cdt1-MCM2-7 Bound to DNA

Jingchuan Sun et al. Nat Struct Mol Biol. .
Free PMC article

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.

Conflict of interest statement

COMPETING FINANCIAL INTERESTS

The authors declare no competing financial interests.

Figures

Figure 1
Figure 1. In vitro assembly of the OCCM complex
(a) Model for Cdc6 recruitment to the replication origin readies the ORC for loading of MCM2-7. (b) Averaged cryo-EM images of the in vitro assembled OCCM. For scale, the box size is 27 nm. An enlarged view with the top area tentatively assigned to ORC-Cdc6 and the lower region to Cdt1-MCM2-7. (c) Mcm2 IP identifies the OCCM components. Using purified ORC, Cdc6, Cdt1, MCM2-7 (lanes 1-4) and origin DNA OCCM was assembled in the presence of ATPγS. OCCM was cleaved off from the plasmid DNA with DNase I and immunoprecipitated with an anti-Mcm2 antibody (lanes 5-7) or with anti-MBP control antibody (lanes 8-10). Asterisk marks nonspecific proteins from antibody-conjugated beads.
Figure 2
Figure 2. Cryo-EM of the eukaryotic OCCM complex
(a) A representative raw cryo-EM image of the purified OCCM complex embedded in vitreous ice. (b) Selected reference-free 2D class averages of the OCCM cryo-EM images (left panel) in comparison with their approximately corresponding reprojections from the 3D reconstruction (right panel). The box size is 34 nm. (c) Surface view of the cryo-EM 3D map of the OCCM complex rendered at the threshold that includes the expected molecular mass of 1.1 MDa. (d) Fourier shell correlation suggests that the 3D map has a resolution of 14 Å. (e) Tilt validation of the cryo-EM 3D map. The predicted tilt axis and tilt angle of each particle pair, based on the cryo-EM map, are plotted as a black dot. Most particle pairs cluster in a region demarcated by the red circle that is centered at the experimental tilt axis (90°) and tilt angle (10°).
Figure 3
Figure 3. Mapping protein and DNA components of the OCCM
(a - f) 2D class averages and 3D reconstruction of OCCM with MBP fused to the C-terminus (CT) of Orc2 (Orc2-MBP) (a), the N-terminus (NT) of Mcm2 (MBP-Mcm2) (b), the NT of Mcm3 (MBP-Mcm3) (c), the CT of Mcm5 (MBP-Mcm5) (d), the NT of Cdt1 (MBP-Cdt1) (e), and the NT of Mcm6 (MBP-Mcm6) (f), respectively. In each left panel, the upper row shows two reference-free class averages, and the lower row shows the same images displayed at a higher contrast level (C=0.3). Each middle panel shows the surface rendered front, back, and bottom views of the 3D map of the MBP-fused OCCM complex. The peripheral MBP density is colored blue. The surface-rendering thresholds were lowered by ~20% to better visualize the small MBP density. Each right panel shows a vertical (a-b) or a horizontal section (c-f) of the 3D map of the MBP-fused OCCM (first column) in comparison to the corresponding section of that of the wild type OCCM (second column). The lower row is displayed at a higher contrast level than the upper row. The red arrows point to the MBP density at the peripheral of OCCM. All MBP fusion complexes were imaged by cryo-EM exception for MBP-Mcm3 (c) that was by negative stained EM. All fusion complexes were cleaved off the plasmid DNA by DNases I except for MBP-Mcm6 that was by Alu I (f). (g) Reference-free class averages of wtOCCM with their plasmid DNA digested either by DNase I (upper row) or by Alu I (lower row). Blue arrows point to dsDNA stub on the top ORC-Cdc6 region of OCCM. (h) MCM2-7 organization as mapped by the four MBP-fused Mcm subunits (red), viewed from the N-terminal end of MCM2-7. Box size is 37 nm in the left panels, 34 nm in the right panels in (a-f), and 31 nm in (g). 3D maps are on same scale.
Figure 4
Figure 4. Segmented cryo-EM structure of the OCCM
(a – d) show four side views of OCCM obtained by consecutive 90°rotations around a vertical axis. (e and f) Top and bottom views by rotating +90° or -90° around the horizontal axis from the side view in (b). (g) Bottom and back side views of the OCCM map shown in semi-transparent surface, docked with homology crystal structure of the Archaeal MCM NTD hexamer (PDB ID: 1LTL). Surface-rendering threshold is set to enclose the expected 1.1-MDa mass of OCCM. (a – g) are on the same scale. Mcm2-7 are abbreviated as M2-7, and Orc1-6 as O1-6, respectively. (h) Ribbon presentation of the Archaeal Orc1/Cdc6 crystal structure, showing the N-terminal peptide meanders away from NT AAA+ domain and joins the middle helical domain (HD) (PDB ID: 2QBY). (i) The segmented Cdc6 density is shown as semi-transparent surface and the rigid-body docked Archaeal Orc1/Cdc6 structure shown as yellow ribbons (PDB ID: 1FNN). The N-terminal peptide shown in blue is at the middle HD region, away from the N-terminal AAA+ domain. The approximate location of N-terminal peptide of Cdc6 in the OCCM structure is labeled with a blue asterisk in (a) and (b), respectively. The C-terminal WHD of Cdc6 contacts the assigned DNA density visible in (e), whereas the N-terminal AAA+ domain reaches down and interacts with the CTD of Mcm3. (j) The Mcm3 density is shown as semi-transparent surface with the rigid-body docked Archaeal Mcm homology structure shown as salmon ribbons (PDB ID: 3F9V).
Figure 5
Figure 5. Cryo-EM structure of the yeast Cdt1-MCM2-7 in the context of the OCCM complex in comparison with the Drosophila MCM2-7 structure
(a) The Cdt1-MCM2-7 density is isolated from the OCCM complex and shown in three surface views. Individual subunits are labeled. (b) The negatively stained EM structure of the Drosophila MCM2-7 in the bottom N-terminal view (EMDB ID: 1134). (c) The ORC-facing C-terminal stereo view of the yeast Cdt1-MCM2-7. Note that the light gray density inside the hexamer chamber resembles DNA. Mcm2-7 are abbreviated as M2-7. (a – c) are on the same scale.
Figure 6
Figure 6. Upon recruitment of Cdt1-MCM2-7, ORC-Cdc6 undergoes concerted conformational changes into a right-handed spiral structure
(a) The ORC-Cdc6-DNA structure alone. (b) The ORC-Cdc6-DNA structure extracted from the OCCM structure. The question mark indicates the tentative assignment of Orc6. (c) Stereo side view of ORC-Cdc6 extracted from the OCCM structure. The dashed black curve traces the right-handed helical rise in the order of Orc3, Orc2, Orc5, Orc4, Orc1, and Cdc6. The vertical rise from the lowest Orc3 to the highest Cdc6 is 34 Å as labeled. The dashed red line illustrates the bound dsDNA. Orc1-6 are abbreviated as O1-6.
Figure 7
Figure 7. The DNA apparently passes through the middle of the OCCM complex
(a) 3D cryo-EM map of OCCM with the positions of horizontal sections labeled from the top 0 to the bottom 7. (b) 2D sections of OCCM with resolved DNA density labeled by the blue arrow. (c) A side view of the OCCM structure with the front Orc3 and Mcm5 densities removed to allow viewing of the interior elongated DNA densities extending from the top to the bottom, as outlined by a pair of dashed red lines. (d) The central axis of MCM2-7 hexamer is tilted 12° away from the helical axis of the spiral ORC-Cdc6 structure. (e) A ribbon representation of the crystal structure of the yeast RFC-PCNA complex (PDB ID: 1SXJ), showing the 9° mismatch between the central axis of the PCNA ring and the spiral axis of the RFC-A-RFC-E pentamer. Note that only the AAA+ motor domains in RFC are helically arranged. Mcm2-7 are abbreviated as M2-7, and Orc1-6 as O1-6, respectively. (a, c, d, e) are on the same scale. The box size in (b) is 22 nm.

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