Cdc6-induced conformational changes in ORC bound to origin DNA revealed by cryo-electron microscopy

Abstract

The eukaryotic origin recognition complex (ORC) interacts with and remodels origins of DNA replication prior to initiation in S phase. Here, we report a single-particle cryo-EM-derived structure of the supramolecular assembly comprising Saccharomyces cerevisiae ORC, the replication initiation factor Cdc6, and double-stranded ARS1 origin DNA in the presence of ATPγS. The six subunits of ORC are arranged as Orc1:Orc4:Orc5:Orc2:Orc3, with Orc6 binding to Orc2. Cdc6 binding changes the conformation of ORC, in particular reorienting the Orc1 N-terminal BAH domain. Segmentation of the 3D map of ORC-Cdc6 on DNA and docking with the crystal structure of the homologous archaeal Orc1/Cdc6 protein suggest an origin DNA binding model in which the DNA tracks along the interior surface of the crescent-like ORC. Thus, ORC bends and wraps the DNA. This model is consistent with the observation that binding of a single Cdc6 extends the ORC footprint on origin DNA from both ends.

Figure 1. The smallest ORC subunit Orc6 interacts with Orc2, but not with Orc3

(A - C) Radiolabeled Orc1/2/3/4/5 (A), or deletion constructs of Orc2 (B, C) were expressed using an in vitro transcription/translation system and pulled down by GST (G) or GST-Orc6 (6). Input (5%) and bound materials (30%) were visualized. V, expression vector; FL, full length. (D) Comparison of reference-free class averages of EM images of ORC and Orc1-5 subcomplex. Image adapted from (Chen et al.). (E) A complete assignment of all six subunits of ORC.

Figure 2. Binding of ORC to origin DNA stabilizes the structure and induces a 20 ° rigid- body rotation at the ORC top lobe comprised of Orc1, Orc4 and Orc5

(A) A raw image of ORC mixed with 66-bp ARS1 dsDNA at 1:1.2 molar ratio. Several individual particles are marked in white circles. (B) Selected 2D reprojections (left) and corresponding reference-based class averages (right) of the ORC-dsDNA complex. (C) Cryo-EM 3D map of ORC alone in top (upper) and front side (lower) views. (D) Cryo-EM 3D map of ORC-dsDNA shown in top (upper) and front side (lower) views. Individual subunits are labeled. See also Fig. S1 and S2 for 3D reconstruction details.

Figure 3. Cdc6 binding onto the ORC-DNA structure induces a rotation of Orc1

(A) Negatively stained EM image averages of ORC-DNA and (B) Cdc6-ORC-DNA. Gain of density in the presence of Cdc6 is marked by filled arrows in (B). Orc1 re-arrangement upon Cdc6 binding is indicated by white arrows. (C) Comparison of cryo-EM 3D map of ORC-DNA with that of ORC-DNA-Cdc6 shown in (D). The precise boundary between Orc2 and Orc3 cannot be determined based on the current data. Orc1 density is painted in light blue, and Orc1 NTD painted in cyan. Cdc6 density in (D) is painted in magenta. Rearrangement of Orc1 density upon Cdc6 binding is illustrated by a pair of blue arrows. The protruding density in ORC-Cdc6-DNA (D) is painted in red and tentatively assigned to be a part of Orc6. See also Fig. S1 and S2 for 3D reconstruction details.

Figure 4. Repression of Orc6 - Orc1^{301 - 400} binding via Orc1 N-terminal domain

Radiolabeled Orc1 (FL) or its deletion constructs were expressed in vitro and pulled down by GST-Orc6 (Panel A and 6 in panel B) or GST (G in panel B). Input (5%) and bound materials (30%) were visualized.

Figure 5. Interpretation of 3D cryo-EM structure of the ORC-DNA- Cdc6 super-assembly

(A) Crystal structure of archaeal Orc1/Cdc6 in the absence of DNA in cartoon view showing the three linearly arranged domains (Liu et al., 2000). (B) Archaeal Orc1 in DNA binding conformation in cartoon view showing the highly curved C-shaped arrangement of the three domains. DNA molecule is not shown for clarity. Two pink ellipses mark the approximately DNA binding regions in the N-terminal α/β-folded ATPase domain and the C-terminal WHD (Dueber et al., 2007). (C) Segmentation of the 3D map. Each subunit is shown in a different color. Segger (Pintilie et al., 2010) as implemented in Chimera (Pettersen et al., 2004) was used to segment the density. Cdc6 at the side, Orc1, Orc4 at the top, and Orc5 in the middle are more separated, their boundary definition is objective. Orc2, Orc3, and Orc6 are tightly packed at the bottom lobe and as such their boundaries are speculative. (D) Docking with the highly curved archaeal Orc1/Cdc6 crystal structure (PDB id 2qby) as individual rigid body for the segmented Orc1-5 densities (Fig. S3). Archaeal Orc1/Cdc6 crystal structure in linear form (PDB id 1fnn) was docked as a rigid body into the segmented yeast Cdc6 density (Fig. S3). No Orc6 homolog crystal structure was found and its density was left undocked. The black double arrow in the front view indicates the distance between the putative Orc4 arginine finger and the Orc1 nucleotide-binding site. See also Fig. S3 for how the rigid-body docked homolog crystal structure fits the segmented individual subunits Orc1, Orc4, Orc5, and Cdc6.

Figure 6. A model for replication origin DNA recognition by ORC and Cdc6

(A) Individual subunits are pulled apart to reveal their C-shaped structures of Orc1 through Orc5. Cdc6 density is extended. Assuming the yeast ORC subunits bind replication DNA in a similar manner, i.e. each subunit bind DNA with two claws, the origin DNA should follow a highly curved path lining the inner surface of the large crescent-shaped structure of ORC. The DNA path is illustrated by the dashed orange band. (B) An atomic model built with 72-bp dsDNA nested at the inner surface of the crescent-like ORC structure. Such DNA recognition model satisfies the general DNA interaction mode, and predicts a highly bent origin DNA onto which the MCM2-7 helicase will be loaded in the following steps. See also Movie S1. See also Fig. S4 for the architectural similarity between ScORC and DmORC.