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, 24 (3), 316-324

Structural Basis of Mcm2-7 Replicative Helicase Loading by ORC-Cdc6 and Cdt1

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Structural Basis of Mcm2-7 Replicative Helicase Loading by ORC-Cdc6 and Cdt1

Zuanning Yuan et al. Nat Struct Mol Biol.

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.

Conflict of interest statement

COMPETING FINANCNCIAL INTNTERESTS

The authors declare no competing financial interests.

Figures

Figure 1
Figure 1. Cryo-EM and overall structure of the S. cerevisiae OCCM complex
(a) A typical motion-corrected raw image of frozen OCCM particles recorded on a direct detector. (b) Selected six 2D averages representing the particles in different views. (c) 3D cryo-EM map of OCCM color coded by local resolution. Overall resolution is 3.9 Å. (d) Cartoon view of the atomic model of OCCM as viewed from Front side. The two black arrows in left indicates that the ORC-Cdc6 ring lays on the MCM ring tilted by an angle of ~17°. The two black arrows in middle shows the DNA in central channel is bent by ~25°. The blue oval marks the short helix of Orc6. (e) Cartoon view of the OCCM model as viewed from the backside. The black circles mark the WHDs of Mcm3, Mcm4, Mcm6 and Mcm7, respectively.
Figure 2
Figure 2. Cross-linking/mass spectrometry analysis of S. cerevisiae OCCM complex
(a) Linkage map showing the observed cross-linked residue pairs within the MCM2-7/Cdt1 complex. Intra-molecular cross-links are color coded, while inter-molecular cross-links are shown in black. (b) Linkage map showing the observed cross-linked residue pairs within the ORC/Cdc6 complex. Intra-molecular cross-links are color coded, while inter-molecular cross-links are shown in black. (c) Linkage map showing the observed cross-linked residue pairs between ORC/Cdc6 complex and Mcm2-7/Cdt1 complex. Orc6, which was only partially resolved by cryo-EM, is in close proximity to Mcm2 and Cdt1. The Winged Helix Domain of Mcm5, that was only partially resolved by cryo-EM, is in close proximity to the N-terminal region of Orc2.
Figure 3
Figure 3. ORC-Cdc6 encircles the origin DNA with the Orc4 insertion helix binding to a major groove
(a) Domain organization of S. cerevisiae Orc1-5 subunits and Cdc6. Dashed lines mark the ORC-Cdc6 core regions resolved in our model (TFIIB and CTD, transcription factor-II-like and C-terminal domains in Orc6; BAH, bromo-adjacent homology domain in Orc1). (b) The ORC-Cdc6 structure in our S. cerevisiae OCCM model in top view. (c) Superposition of Orc1-5 and Cdc6, highlighting their similar overall structures. Orc2 lacked the AAA-lid domain, resulting in a relatively open interface between Orc2-Cdc6. Orc3 had an insertion domain between the AAA-lid domain and the WHD domain that interacted with Orc6. The blue arrows point to structures with which these marked elements interact. The black arrow points to the missing lid domain in Orc2. (d) Crystal structure of Drosophila ORC complex in a similar subunit color scheme. (e, f) Alignment of DmORC with ScORC-Cdc6 using the most similar Orc3-5 region as a reference showed that the AAA-RecA-fold domain of DmOrc1 (e) and WHD of DmOrc2 (f) needed to move and rotate by 180° to assume their respective position in ScOCCM. See also Supplemental video 1.
Figure 4
Figure 4. Nucleotide binding sites and configuration in OCCM
(a) Cut-open top view of ORC-Cdc6 and Mcm2-7 shown in surface view. The four ATPγS molecules identified in ORC-Cdc6 at the interface between Cdc6-Orc1, Orc1-Orc4, Orc4-Orc5, and Orc5-Orc3 (right), and four ATPγS molecules in Mcm2-7 at the interface between Mcm2-Mcm6, Mcm6-Mcm4, Mcm4-Mcm7, and Mcm7-Mcm3 are shown as spheres (carbon in green, oxygen in red, nitrogen in blue, sulfur in yellow). (b) The positons of the observed nucleotides in OCCM relative to the DNA, which is shown in orange surface. The left panel is a side view with Mcm4 in front and the right panel is a top view with ORC-Cdc6 on top, but proteins are not shown in order to highlight the nucleotides. The four ATPγS molecules in ORC-Cdc6 are co-planar, but the plane is tilted by ~17° with respect to the plane formed by the nucleotides in Mcm2-7. An imaginary circle defined by the nucleotide in ORC-Cdc6 is larger (75 Å) than the circle defined by nucleotides in Mcm2-7 (65 Å), and the two circles are acentric.
Figure 5
Figure 5. Extensive interactions between Cdt1 and MCM hexamer
(a) OCCM structure with Cdt1 electron density shown in blue mesh. The CTD of Cdt1 locates between Mcm6 and Mcm4, over 60 Å away from the NTD and MHD of Cdt1. (b) Zoomed view of the Cdt1 NTD and MHD showing their interactions with Mcm2 and Mcm6. (c) Zoomed view showing the Cdt1 CTD interacting with Mcm6 WHD. The dotted blue line in (a-c) indicates a flexible loop connecting Cdt1 MHD and CTD. (d) The top view (left) and front side view (right) of Mcm2-7 structure in cartoon and semi-transparent surface view. The red oval marks Mcm6-WHD in OCCM, and the dashed red oval the position of Mcm6-WHD in CMG helicase. The blue arrow shows the displacement of Mcm6 WHD in OCCM due to interaction with Cdt1 CTD. Such displacement forms an unobstructed Mcm2-7 C-terminal face for binding with ORC-Cdc6.
Figure 6
Figure 6. Conformational changes between the Mcm2-7 in OCCM and double-hexamer (D-H)
(a) Comparison of the top CTD view (left) and the bottom NTD view (right) of the Mcm2-7 structure in the double-hexamer (gray cartoon) with the Mcm2-7 structure in the OCCM. The two structures were aligned using CTDs of Mcm4-6-7 as reference. Changes in the CTD ring are focused in Mcm2-5-3. The NTD ring rotated en bloc by about 25°. (b) Front Mcm2/5 side view of the Mcm2-7 hexamer in the OCCM structure (left) as compared to that in the D-H (right). Transitioning from OCCM to double hexamer, each CTD AAA+ domain and NTD of Mcm2 and Mcm5 undergoes a combination of rotation and translation, with the degree of rotation and translation shown as labeled. Mcm5 NTD needs to rotate by as much as 50° to close the DNA entry gate. (c) A sketch showing how the gate between Mcm2 and Mcm5 can be open for DNA insertion in OCCM (left) and how the gate is closed in the D-H (right).
Figure 7
Figure 7. Interactions between OCCM and DNA
(a) An overview of OCCM-DNA structure in side view. Subunits in front of DNA including parts of Orc1 and Cdc6, and all of Mcm3 and Mcm5 are removed to show DNA. Five rectangle-marked areas are enlarged in panels (c-f). (b-c) Detailed view of the Cdc6 interaction with dsDNA. (d) Orc2 interaction with DNA. (e) Orc4 interaction with DNA. (f) Mcm2-6-4-7 and DNA interfaces. PS1: Pres-sensor 1 β-hairpin loop, H2I: Helix-2-insert β-hairpin loop.

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