| Abstrakt: |
ABSTRACTPreviously we have shown that the establishment of anoriPreplicon 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 oriPreplicons, we monitored the replication of oriPplasmid 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 oriPreplicated but were lost more rapidly from cells than were oriPplasmids, demonstrating that the family of repeats (FR) of oriPacts in cisto 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 oriPreplicated but were lost at a rate of approximately 30% per cell generation in 293/EBNA-1 cells, indicating that the FR inhibitsoriP's establishment in this cell line. FR's enhancement of transcription of a promoter in cisand FR's ability to inhibit replication fork movement do not account solely fororiP'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 oriPderivative in 293/EBNA-1 cells. |
