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Previously we have shown that the establishment of an oriP replicon is dependent on its epigenetic modification, which occurs in only 1 to 10% of proliferating cells (E. R. Leight and B. Sugden, Mol. Cell. Biol. 21:4149-4161, 2001). To gain insights into the cis-acting requirements for the establishment of oriP replicons, we monitored the replication of oriP plasmid derivatives for several weeks following their introduction into cells. In EBNA-1-positive 143B and H1299 cells, plasmids containing only the region of dyad symmetry (DS) of oriP replicated but were lost more rapidly from cells than were oriP plasmids, demonstrating that the family of repeats (FR) of oriP acts in cis to stimulate replication in these cells. Unexpectedly, we found that the DS plasmid was established efficiently in 293/EBNA-1 cells, being lost at a rate of only 8% per cell generation over 24 days posttransfection. However, plasmids containing the FR in addition to the DS of oriP replicated but were lost at a rate of approximately 30% per cell generation in 293/EBNA-1 cells, indicating that the FR inhibits oriP's establishment in this cell line. FR's enhancement of transcription of a promoter in cis and FR's ability to inhibit replication fork movement do not account solely for oriP's inefficient establishment. In addition, DNA looping between FR and DS neither stimulates nor inhibits replication. Deletion of 11 EBNA-1 binding sites in the FR or replacement of the FR with DS sequences, however, does overcome the inhibitory activity of the FR, thereby allowing efficient establishment of the oriP derivative in 293/EBNA-1 cells.

The cis -Acting Family of Repeats Can Inhibit as well as Stimulate Establishment of an oriP Replicon