Abstract
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.
Highlights
Epstein-Barr virus (EBV) is a gammaherpesvirus that causes infectious mononucleosis [12, 42] and is associated with several malignancies, including Hodgkin’s disease, gastric carcinoma, and B-cell lymphomas in immunocompromised individuals [9, 18, 23, 26, 51]
Equimolar amounts of a test plasmid and prokaryotic backbone plasmid were introduced into 143B cells with or without an expression plasmid for EBNA-1 or into C33A/EBNA-1, H1299/1728#3, and 293/EBNA-1 cells that stably express EBNA-1
Given that the family of repeats (FR)’s enhancement of transcription from the our studies contains both replicative (oriP) replicon does not underlie the replicon’s precipitous loss, we addressed whether the FR, by acting as a potent replication fork barrier (RFB), promotes this inefficient establishment. oriP plasmids that initiate DNA synthesis during the last 3 h of S-phase may not be completely synthesized by the end of S-phase, as 3 or more hours are devoted to DNA synthesis through the FR [15]
Summary
Epstein-Barr virus (EBV) is a gammaherpesvirus that causes infectious mononucleosis [12, 42] and is associated with several malignancies, including Hodgkin’s disease, gastric carcinoma, and B-cell lymphomas in immunocompromised individuals [9, 18, 23, 26, 51]. Plasmids containing oriP support efficient replication in EBNA-1-positive cells selected to retain them, being lost at a rate of 2 to 4% per cell generation after removal of selection [29, 56], a rate of loss which resembles that of ARS/CEN plasmids in Saccharomyces cerevisiae [33]. We refer to these plasmids as “established” replicons in that they support efficient DNA synthesis and partitioning each cell cycle. We report that cell lines differ in their ability to support the replication of
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