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First published online September 2, 2003
doi: 10.1242/10.1242/jcs.00708
Research Article |
1 Department of Gene Vectors, GSF-National Research Center for Environment and
Health, Marchioninistrasse 25, 81377 München, Germany
2 Institute for Immunology, GSF-National Research Center for Environment and
Health, Marchioninistrasse 25, 81377 München, Germany
* Author for correspondence (e-mail: schepers{at}gsf.de)
Accepted 11 June 2003
The sequential binding of the origin recognition complex (ORC), Cdc6p and the minichromosome maintenance proteins (MCM2-7) mediates replication competence at eukaryotic origins of DNA replication. The latent origin of Epstein-Barr virus, oriP, is a viral origin known to recruit ORC. OriP also binds EBNA1, a virally encoded protein that lacks any activity predicted to be required for replication initiation. Here, we used chromatin immunoprecipitation and chromatin binding to compare the cell-cycle-dependent binding of pre-RC components and EBNA1 to oriP and to global cellular chromatin. Prereplicative-complex components such as the Mcm2p-Mcm7p proteins and HsOrc1p are regulated in a cell-cycle-dependent fashion, whereas other HsOrc subunits and EBNA1 remain constantly bound. In addition, HsOrc1p becomes sensitive to the 26S proteasome after release from DNA during S phase. These results show that the complex protein-DNA dynamics at the viral oriP are synchronized with the cell division cycle. Chromatin-binding and chromatin-immunoprecipitation experiments on G0 arrested cells indicated that the ORC core complex (ORC2-5) and EBNA1 remain bound to chromatin and oriP. HsOrc6p and the MCM2-7 complex are released in resting cells. HsOrc1p is partly liberated from chromatin. Our data suggest that origins remain marked in resting cells by the ORC core complex to ensure a rapid and regulated reentry into the cell cycle. These findings indicate that HsOrc is a dynamic complex and that its DNA binding activity is regulated differently in the various stages of the cell cycle.
Key words: oriP, Replication, Cell cycle, EBNA1, ORC
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