Chen, Z. Q., Speck, C., Wendel, P., Tang, C. Y., Stillman, B., Lill, H. L. (July 2008) The architecture of the DNA replication origin recognition complex in Saccharomyces cerevisiae. Proceedings of the National Academy of Sciences of the United States of America, 105 (30). pp. 10326-10331. ISSN 0027-8424
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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.
| Item Type: | Paper |
|---|---|
| Uncontrolled Keywords: | STRUCTURAL BASIS ELECTRON-MICROSCOPY CONFORMATIONAL-CHANGES NUCLEOTIDE-BINDING ATPASE ACTIVITY PROTEIN ORC CDC6 VISUALIZATION LOCALIZATION |
| Subjects: | bioinformatics > genomics and proteomics > genetics & nucleic acid processing > DNA, RNA structure, function, modification > DNA replication organism description > yeast > Saccharomyces Investigative techniques and equipment > microscopy > electron microscopy bioinformatics > genomics and proteomics > genetics & nucleic acid processing > protein structure, function, modification > protein types > enzymes Investigative techniques and equipment > optical devices > microscope techniques or equipment |
| CSHL Authors: | |
| Communities: | CSHL labs > Stillman lab |
| Highlight: | Stillman, Bruce W. |
| Depositing User: | Tom Adams |
| Date: | 29 July 2008 |
| Date Deposited: | 25 Aug 2011 19:02 |
| Last Modified: | 20 Jun 2017 16:40 |
| PMCID: | PMC2480615 |
| Related URLs: | |
| URI: | https://repository.cshl.edu/id/eprint/7706 |
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