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Papers In Press, published online ahead of print April 25, 2001
Molecular Biology and Genetics, Johns Hopkins University, Baltimore, MD 21205
Corresponding Author: tkelly{at}jhmi.edu
The six-subunit origin recognition complex (ORC) was originally identified in the yeast Saccharomyces cerevisiae. Yeast ORC binds specifically to origins of replication and serves as a platform for the assembly of additional initiation factors, such as Cdc6 and the Mcm proteins. Human homologues of all six ORC subunits have been identified by sequence similarity to their yeast counterparts, but little is known about the biochemical characteristics of human ORC (HsORC). We have extracted HsORC from HeLa cell chromatin and probed its subunit composition using specific antibodies. The endogenous HsORC, identified in these experiments, contained homologues of Orc1-Orc5, but lacked a putative homologue of Orc6. By expressing HsORC subunits in insect cells using the baculovirus system, we were able to identify a complex containing all six subunits. To explore the subunit-subunit interactions that are required for the assembly of HsORC, we carried out extensive co-immunoprecipitation experiments with recombinant ORC subunits expressed in different combinations. These studies revealed the following binary interactions: HsOrc2-HsOrc3, HsOrc2-HsOrc4, HsOrc3-HsOrc4, HsOrc2-HsOrc6, and HsOrc3-HsOrc6. HsOrc5 did not form stable binary complexes with any other HsORC subunit, but interacted with sub-complexes containing any two of subunits HsOrc2, HsOrc3, or HsOrc4. Complex formation by HsOrc1 required the presence of the HsOrc2, HsOrc3, HsOrc4 and HsOrc5 subunits. These results suggest that the subunits HsOrc2, HsOrc3 and HsOrc4 form a core upon which the ordered assembly of HsOrc5 and HsOrc1 takes place. The characterization of HsORC should facilitate the identification of human origins of DNA replication.
J. Biol. Chem, 10.1074/jbc.M102493200
Submitted on March 20, 2001
Revised on April 25, 2001
Accepted on April 24, 2001
Assembly of the human origin recognition complex
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