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 single particle cryo-EM-derived structure of the supra-molecular assembly comprising of S. cerevisiae ORC, the replication initiation factor Cdc6 and double strand 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, particularly re-orientating 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.

Genome-wide chromatin footprinting reveals changes in replication origin architecture induced by pre-RC assembly

“…An extension of this G2 ORC footprint in G1 suggested that pre-RC assembly alters the protein-DNA occupancy at the origin (Diffley et al 1994;Perkins and Diffley 1998;Speck et al 2005). Consistent with this change in the molecular architecture of the origin, cryo-electron microscopy (cryo-EM) studies have also revealed a dramatic Cdc6-induced change in the conformation of ORC on template DNA (Sun et al 2012). In vitro pre-RC assembly experiments demonstrated the capability of loading multiple Mcm2-7 double hexamers that can passively translocate along template DNA prior to activation in S phase (Bowers et al 2004;Evrin et al 2009;).…”

“…Previous in vitro DNase I hypersensitivity mapping at individual origins estimated a similar ORC footprint expansion induced by Cdc6 binding (Speck et al 2005). A recent ORC-Cdc6 cryo-EM study suggested that ORC adopts a crescent structure along the DNA, with Cdc6 bridging the gap between the ends of the crescent where DNA both enters and exits the ORC-Cdc6 structure (Sun et al 2012). We propose that the ORC-Cdc6 footprint extension direction is driven by chromatin or sequence-specific interactions that influence MNase accessibility to the DNA at the interface of the ORC-Cdc6 structure.…”

Structural and mechanistic insights into Mcm2–7 double-hexamer assembly and function

“…It is therefore not feasible to obtain homogeneous OCM preparations for 3D reconstruction. Because we know the 3D EM structures of ORC-Cdc6 and the OCCM complexes (Sun et al 2012(Sun et al , 2013, we are able to interpret the 2D structure of the OCM with confidence.…”

Helicase Loading at Chromosomal Origins of Replication

“…The latter hypothesis would explain why ORC does not bind either Cdt1 or Mcm2 -7 in the absence of Cdc6 (Takara and Bell 2011) but isolated Orc6 can bind Cdt1 (Chen et al 2007). Consistent with this model, recent EM studies of ORC bound to DNA in the presence and absence of Cdc6 suggested that Orc6 becomes exposed upon Cdc6 binding (Sun et al 2012). …”

“…Studies in Xenopus extracts have shown that Cdt1 associates with chromatin in the absence of Cdc6 (Gillespie et al 2001;Tsuyama et al 2005); however, only Cdt1 associated in the presence of Cdc6 is able to contribute to Mcm2 -7 loading (Tsuyama et al 2005). Consistent with a robust interaction between Cdc6 and ORC, a complex between the proteins has been structurally characterized (Speck et al 2005;Sun et al 2012) and origin-bound ORC stimulates Cdc6 ATP hydrolysis (Randell et al 2006). …”