Two domains of the Epstein-Barr virus origin DNA-binding protein, EBNA1, orchestrate sequence-specific DNA binding

The EBNA1 (for Epstein-Barr nuclear antigen 1) protein of Epstein-Barr viru s governs the replication and partitioning of the viral genomes during late nt infection by binding to specific recognition sites in the viral origin o f DNA replication. The crystal structure of the DNA binding portion of the EBNA1 protein revealed that this region comprises two structural motifs; a core domain, which mediates protein dimerization and is structurally homolo gous to the DNA binding domain of the papillomavirus E2 protein, and a flan king domain, which mediated all the observed sequence-specific contacts. To test the possibility that the EBNA1 core domain plays a role in sequence-s pecific DNA binding not revealed in the crystal structure, we examined the effects of point mutations in potential hydrogen bond donors located in an cu-helix of the EBNA1 core domain whose structural homologue in E2 mediates sequence-specific DNA binding. We show that these mutations severely reduc e the affinity of EBNA1 for its recognition site, and that the core domain, when expressed in the absence of the flanking domain, has sequence-specifi c DNA binding activity. Flanking domain residues were also found to contrib ute to the DNA binding activity of EBNA1, Thus, both the core and flanking domains of EBNA1 play direct roles in DNA recognition.