Reconstitution of Epstein-Barr virus-based plasmid partitioning in buddingyeast

The EBNA1 protein of Epstein-Barr virus (EBV) mediates the partitioning of EBV episomes and EBV-based plasmids during cell division by a mechanism tha t appears to involve binding to the cellular EBP2 protein on human chromoso mes. We have investigated the ability of EBNA1 and the EBV segregation elem ent (FR) to mediate plasmid partitioning in Saccharomyces cerevisiae. EBNA1 expression alone did not enable the stable segregation of FR-containing pl asmids in yeast, but segregation was rescued by human EBP2, The reconstitut ed segregation system required EBNA1, human EBP2 and the FR element, and fu nctionally replaced a CEN element, An EBP2: binding mutant of EBNA1 and an EBNA1 binding mutant of EBP2 each failed to support FR-plasmid partitioning , indicating that an EBNA1-EBP2 interaction is required, The results provid e direct evidence of the role of hEBP2 in EBNA1-mediated segregation and de monstrate that heterologous segregation systems can be reconstituted in yea st.