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

Abstract

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/.

We thank C. Fox and P. Lambert for help with two-dimensional gel electrophoresis, as well as S. Harkins-Perry and S. Bartley for extensive technical assistance on the ChIP. We thank P. Hua for the initial construction of 8xRep*, S. Adhya for the pBend2 vector, J. Hearing for pHEBO1.1 dpm 3+4 vector, S. Duellman for wtEBNA1, A. Schepers for antibodies to ORC2/3 and EBNA1, and R. R. Burgess and N. Thompson for antibodies to the Softag1 epitope. We thank Chen-Yu Wang for providing her findings on surveying Raji ori for EBNA1-binding sites by EMSA. Finally, we thank J. Yates and members of the Sugden laboratory for their critical reviews of the manuscript.

This study was supported by Public Health Service grants CA-22443, P30-CA-014520, and T32-CA-009135. B.S. is an American Cancer Society Research Professor.