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.
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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%) an...
Figure 2
Binding of ORC to origin DNA stabilizes the structure and induces a 20 u00b0 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-base...
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 ...
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 visual...
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 car...
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...
Figure images are served from the NIH/NLM PubMed Central Open Access Subset or Europe PMC; copyright remains with the publishers and authors.
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