An analysis of essential elements of licensed, plasmid DNA replication in mammalian cells: Studies of natural and artificial latent origins of Epstein-Barr virus

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 EBNA1-binding sites both necessary and sufficient in cis for its origin activity. These sites have an essential 21bp center-to-center spacing, are bent by EBNA1, and recruit ORC. The properties shared between DS and Rep* define cis and trans characteristics of a mammalian, extrachromosomal replicon.; This comparison of two separate, EBNA1-dependent origins prompted us to identify which essential characteristics of these minimal origins contribute to the efficiency of initiating DNA synthesis. Because a pair of EBNA1-binding sites was determined to be the minimal cis-acting element required for origin function, we sought to characterize further the effect that alterations to these binding sites had upon the efficiency of the initiation of DNA synthesis. To this end, we constructed several artificial, EBNA1-dependent origins of DNA synthesis containing EBNA1-binding sites having a >25-fold range of affinities. Using these engineered origins, we have found a direct correlation between the affinity of EBNA1 binding to an origin and both the efficiency of initiating DNA synthesis and of supporting plasmid establishment with that origin. In addition, the presence of TRF2-binding sites flanking the pairs of EBNA1-binding sites enhanced the replicative efficiency of these origins. One of these engineered origins, composed of four EBNA1-binding sites from FR flanked by binding sites for TRF2, initiated DNA synthesis and supported the formation of colonies more efficiently than did wtDS. This finding indicates that EBV has been selected to evolve and retain a sub-optimal origin of DNA synthesis.