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

http://www.ncbi.nlm.nih.gov/pubmed/18647841

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.

Pubmed ID: 18647841 RIS Download

Mesh terms: Carrier Proteins | Cell Cycle Proteins | DNA Replication | DNA, Fungal | Image Processing, Computer-Assisted | Imaging, Three-Dimensional | Immunoprecipitation | Maltose-Binding Proteins | Microscopy, Electron | Models, Molecular | Molecular Conformation | Protein Structure, Tertiary | Replication Origin | Saccharomyces cerevisiae | Saccharomyces cerevisiae Proteins

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Associated grants

  • Agency: NIGMS NIH HHS, Id: GM45436
  • Agency: NIGMS NIH HHS, Id: GM74985
  • Agency: Medical Research Council, Id: MC_U120085811
  • Agency: NCI NIH HHS, Id: P01 CA013106
  • Agency: NCI NIH HHS, Id: P01 CA013106-310025
  • Agency: NCI NIH HHS, Id: P01 CA013106-38S10025
  • Agency: NIGMS NIH HHS, Id: R01 GM045436
  • Agency: NIGMS NIH HHS, Id: R01 GM045436-20
  • Agency: NIGMS NIH HHS, Id: R01 GM074985

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