The linking regions of EBNA1 are essential for its support of replication and transcription

The ability of distant cis-acting DNA elements to interact functionally has been proposed to be mediated by the interaction of proteins associated sit e specifically with those cis-acting elements. We have found that the DNA-l inking regions of EBNA1 are essential for its contribution to both replicat ion and transcription. The synthesis of plasmids containing the Epstein-Bar r virus (EBV) origin of plasmid replication (oriP) can be mediated entirely by the cellular machinery; however, the replicated molecules are lost rapi dly from proliferating cells. When EBNA1 is provided in trans, plasmids con taining oriP (oriP plasmids) are synthesized during repeated S phases, and the newly formed daughter molecules are precisely segregated to the daughte r cells. The contribution(s) of EBNA1 to the stable replication of oriP pla smids is therefore likely to be postsynthetic. In latently infected cells, EBNA1 also regulates the expression of multiple EBV promoters located as ma ny as 10 kbp away. EBNA1 supports replication and transcription through bin ding to oriP; both the ability of EBNA1 to bind to DNA and the integrity of its binding sites in oriP are required. However, DNA binding by EBNA1 is n ot sufficient to support replication or transcription, indicating that an a dditional activity (or activities) is required. EBNA1 links DNAs to which i t binds and can form a loop between the two subelements of oriP, the family of repeats and the region of dyad symmetry, each of which contains multipl e binding sites for EBNA1. We have constructed a set of derivatives of EBNA 1 which contain both, one, or neither of its linking regions in various con texts. Analyses of these derivatives demonstrate that the linking regions o f EBNA1 are essential for its support of replication and transcription and that the ability of derivatives of EBNA1 to link DNAs correlates strongly w ith their support of these activities in cells. These findings indicate tha t protein-protein associations of the linking regions of EBNA1 underlie its long-range contributions to replication and transcription.