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Identifying a Property of Origins of DNA Synthesis Required to Support Plasmids Stably in Human Cells

Chen-Yu Wang and Bill Sugden
Proceedings of the National Academy of Sciences of the United States of America
Vol. 105, No. 28 (Jul. 15, 2008), pp. 9639-9644
Stable URL: http://www.jstor.org/stable/25463026
Page Count: 6
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Since scans are not currently available to screen readers, please contact JSTOR User Support for access. We'll provide a PDF copy for your screen reader.
Identifying a Property of Origins of DNA Synthesis Required to Support Plasmids Stably in Human Cells
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Abstract

The plasmid origin of replication, oriP, of Epstein-Barr Virus (EBV) was identified in an assay to detect autonomously replicating sequences (ARSs) in human cells. Raji ori, a second origin in EBV, functions in vivo but fails in long-term ARS assays. We examined the initiating element, DS, within oriP and Raji ori to resolve this paradox. DS, but not Raji ori, binds EBNA1; whereas both act as ARSs in short-term assays, with DS being more efficient, only DS can act as an ARS in long-term assays. Surprisingly, we found that DS supported the establishment of a plasmid with Raji ori in cis and that after deletion of DS, Raji ori could now act as an ARS in the long term. This finding explains the frequent failure of ARS assays in mammalian cells. More origins can initially act as ARSs than can be established. We identified one requirement for ARSs to be established: They must function efficiently enough initially to generate a wide distribution of numbers of plasmids per cell. Only the cells that have more than a threshold number of plasmids can survive selections imposed on the cells to retain these replicons.

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