Essential elements of a licensed, mammalian plasmid origin of DNA synthesis.
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.
Wang
Jindong
J
McArdle Laboratory for Cancer Research, University of Wisconsin--Madison, Madison, WI 53706, USA.
Lindner
Scott E
SE
Leight
Elizabeth R
ER
Sugden
Bill
B
D001483:Base Sequence; D001665:Binding Sites; D002478:Cells, Cultured; D004261:DNA Replication; D019309:Epstein-Barr Virus Nuclear Antigens / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics / Q000378:metabolism*; D018741:Replication Origin
D019309:Epstein-Barr Virus Nuclear Antigens