title: The architecture of the DNA replication origin recognition complex in Saccharomyces cerevisiae.
abstract: 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.
authors.affiliation: Biology Department, Brookhaven National Laboratory, Upton, NY 11973, USA.
authors.forename: Zhiqiang
authors.initials: Z
authors.lastname: Chen
authors.forename: Christian
authors.initials: C
authors.lastname: Speck
authors.forename: Patricia
authors.initials: P
authors.lastname: Wendel
authors.forename: Chunyan
authors.initials: C
authors.lastname: Tang
authors.forename: Bruce
authors.initials: B
authors.lastname: Stillman
authors.forename: Huilin
authors.initials: H
authors.lastname: Li
mesh_terms: D002352:Carrier Proteins / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000235:genetics / Q000502:physiology; D004261:DNA Replication; D004271:DNA, Fungal / Q000737:chemistry*; D007091:Image Processing, Computer-Assisted; D021621:Imaging, Three-Dimensional; D047468:Immunoprecipitation; D058619:Maltose-Binding Proteins; D008854:Microscopy, Electron / Q000379:methods; D008958:Models, Molecular; D008968:Molecular Conformation; D017434:Protein Structure, Tertiary; D018741:Replication Origin; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000502:physiology
chemical_list: C066503:CDC6 protein, S cerevisiae; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D004271:DNA, Fungal; D058619:Maltose-Binding Proteins; D029701:Saccharomyces cerevisiae Proteins