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Molecular and Cellular Biology, February 2006, p. 1124-1134, Vol. 26, No. 3
0270-7306/06/$08.00+0     doi:10.1128/MCB.26.3.1124-1134.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Essential Elements of a Licensed, Mammalian Plasmid Origin of DNA Synthesis

Jindong Wang, Scott E. Lindner, Elizabeth R. Leight, and Bill Sugden^*

McArdle Laboratory for Cancer Research, University of Wisconsin—Madison, Madison, Wisconsin 53706

Received 22 August 2005/ Returned for modification 17 October 2005/ Accepted 9 November 2005

We developed a mammalian plasmid replicon with a formerly uncharacterized origin of DNA synthesis, 8xRep*. 8xRep* functions efficiently to support once-per-cell-cycle synthesis of plasmid DNA which initiates within Rep*. By characterizing Rep*'s requirements for acting as an origin, we have uncovered several striking properties it shares with DS, the only other well-characterized, licensed, mammalian plasmid origin of DNA synthesis. Rep* contains a pair of previously unrecognized Epstein-Barr nuclear antigen 1 (EBNA1)-binding sites that are both necessary and sufficient in cis for its origin activity. These sites have an essential 21-bp center-to-center spacing, are bent by EBNA1, and recruit the origin recognition complex. The properties shared between DS and Rep* define cis and trans characteristics of a mammalian, extrachromosomal replicon. The role of EBNA1 likely reflects its evolution from cellular factors involved in the assembly of the initiation machinery.

Supplemental material for this article may be found at http://mcb.asm.org/.

J.W. and S.E.L. contributed equally to this study.

Present address: Department of Molecular Biology and Pharmacology, Washington University School of Medicine, St. Louis, MO 63110.

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