Nucleoprotein complexes and DNA 5'-ends at oriP of Epstein-Barr virus

Understanding protein-DNA interactions in vivo at origins of DNA replication throughout the cell cycle may shed further insight on the mechanisms of initiation and replication control. The Burkitt's lymphoma cell line Raji harbors multiple copies of latent Epstein-Barr virus. Once per cell cycle the origin of plasmid replication of Epstein-Barr virus provides replication function in cis for the viral DNA. Here we examined in vivo nucleoprotein complexes on the initiator element of the origin before and after DNA synthesis. For this purpose Raji cells were synchronously growth arrested in G1 phase by mimosine and in mitosis by colchicine, respectively. The association of the initiator element with proteins was visualized by footprinting with dimethyl sulfate and ligation mediated polymerase chain reaction. Methylation patterns indicated a novel binding activity within each element of a nonamer repeated three times at the initiator element. This activity was strongly diminished in mitotic cells. Furthermore, 5'-ends of Epstein-Barr virus DNA were mapped to the nonamers by ligation mediated polymerase chain reaction, suggesting potential initiation sites for replication from DS.