The architecture of the DNA replication origin recognition complex in Saccharomyces cerevisiae

Biology Department, Brookhaven National Laboratory, Upton, NY 11973, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 07/2008; 105(30):10326-31. DOI: 10.1073/pnas.0803829105
Source: PubMed


The origin recognition complex (ORC) is conserved in all eukaryotes. The six proteins of the Saccharomyces cerevisiae ORC that form a stable complex bind to origins of DNA replication and recruit prereplicative complex (pre-RC) proteins, one of which is Cdc6. To further understand the function of ORC we recently determined by single-particle reconstruction of electron micrographs a low-resolution, 3D structure of S. cerevisiae ORC and the ORC-Cdc6 complex. In this article, the spatial arrangement of the ORC subunits within the ORC structure is described. In one approach, a maltose binding protein (MBP) was systematically fused to the N or the C termini of the five largest ORC subunits, one subunit at a time, generating 10 MBP-fused ORCs, and the MBP density was localized in the averaged, 2D EM images of the MBP-fused ORC particles. Determining the Orc1-5 structure and comparing it with the native ORC structure localized the Orc6 subunit near Orc2 and Orc3. Finally, subunit-subunit interactions were determined by immunoprecipitation of ORC subunits synthesized in vitro. Based on the derived ORC architecture and existing structures of archaeal Orc1-DNA structures, we propose a model for ORC and suggest how ORC interacts with origin DNA and Cdc6. The studies provide a basis for understanding the overall structure of the pre-RC.

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    • "GST-Orc6 was cloned and overexpressed using the pET system (Novagen). Template DNAs for in vitro transcription/translation of ORC subunits were derived from pCITE-2a(+) (Novagen) carrying individual full-length ORC genes (Chen et al., 2008); templates for deletion constructs were prepared using the QuikChange Site-Directed Mutagenesis Kit (Stratagene) or PCR. The TNT T7 Quick system for PCR DNA (Promega) was used for transcription/translation when the PCR-amplified DNAs were templates. "
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    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.
    Full-text · Article · Mar 2012 · Structure
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    • "Eukaryotic origins, on the other hand, tend to be very loosely defined, with little or no consensus origin sequences. Saccharomyces cerevisiae has the bestcharacterized origins, each containing a single essential 17 bp ARS consensus sequence and a nearby adenine-rich region (Breier et al., 2004; Chen et al., 2008). In meta-zoans, origins do not share any known consensus sequence, and mapping typically cannot narrow down replication start sites to less than a few kb (Gilbert, 2001; DePamphilis, 2005). "
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    ABSTRACT: Replication initiation is a key event in the cell cycle of all organisms and oriC, the replication origin in Escherichia coli, serves as the prototypical model for this process. The minimal sequence required for oriC function was originally determined entirely from plasmid studies using cloned origin fragments, which have previously been shown to differ dramatically in sequence requirement from the chromosome. Using an in vivo recombineering strategy to exchange wt oriCs for mutated ones regardless of whether they are functional origins or not, we have determined the minimal origin sequence that will support chromosome replication. Nearly the entire right half of oriC could be deleted without loss of origin function, demanding a reassessment of existing models for initiation. Cells carrying the new DnaA box-depleted 163 bp minimal oriC exhibited little or no loss of fitness under slow-growth conditions, but were sensitive to rich medium, suggesting that the dense packing of initiator binding sites that is a hallmark of prokaryotic origins, has likely evolved to support the increased demands of multi-forked replication.
    Preview · Article · Oct 2009 · Molecular Microbiology
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