Complex protein-DNA dynamics at the latent origin of DNA replication of Epstein-Barr virus.
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.
Ritzi
Marion
M
Department of Gene Vectors, GSF-National Research Center for Environment and Health, Marchioninistrasse 25, 81377 München, Germany.
Tillack
Kristina
K
Gerhardt
Jeannine
J
Ott
Elisabeth
E
Humme
Sibille
S
Kremmer
Elisabeth
E
Hammerschmidt
Wolfgang
W
Schepers
Aloys
A
D000818:Animals; D002453:Cell Cycle; D002478:Cells, Cultured; D002843:Chromatin / Q000378:metabolism* / Q000821:virology; D004268:DNA-Binding Proteins / Q000378:metabolism*; D019309:Epstein-Barr Virus Nuclear Antigens / Q000378:metabolism*; D005434:Flow Cytometry; D004854:Herpesvirus 4, Human / Q000502:physiology*; D006801:Humans; D051738:Origin Recognition Complex; D010957:Plasmids; D011485:Protein Binding; D016192:Resting Phase, Cell Cycle / Q000502:physiology; D016196:S Phase / Q000502:physiology; D014764:Viral Proteins / Q000378:metabolism; D014779:Virus Replication / Q000502:physiology
D002843:Chromatin; D004268:DNA-Binding Proteins; D019309:Epstein-Barr Virus Nuclear Antigens; C497224:ORC2 protein, human; D051738:Origin Recognition Complex; D014764:Viral Proteins; C417029:EBV-encoded nuclear antigen 1