SEPARATION OF THE COMPLEX DNA-BINDING DOMAIN OF EBNA-1 INTO DNA RECOGNITION AND DIMERIZATION SUBDOMAINS OF NOVEL STRUCTURE

EBNA-1 is essential for replication of the latent episomal form of the Epstein-Barr virus genome and is involved in regulation of viral late ncy promoters. EBNA-1 activity is mediated through direct DNA binding. The DNA binding and dimerization functions of EBNA-1 have previously been located to a carboxy-terminal domain, amino acids (aa) 459 to 607 . To identify and define the subdomains for these two functions, we cr eated an extensive series of deletions and point mutations in an EBNA- 1 (aa 408 to 641) background. The ability of the EBNA-1 mutants to het erodimerize with a wild-type EBNA-1 (aa 459 to 641) polypeptide was te sted in immunoprecipitation assays with a monoclonal antibody, EBNA.OT 1x, that recognizes EBNA-1 (aa 408 to 641) but not EBNA-1 (aa 459 to 6 41). These experiments revealed that mutations affecting dimerization occurred over two separate regions, aa 501 to 532 and aa 554 to 598. D NA binding was tested in mobility shift assays against a panel of olig onucleotide-binding sites. Dimerization was a prerequisite for DNA bin ding. The DNA recognition domain was localized to a separate region, a a 459 to 487, upstream of the dimerization domain. EBNA-1 variants car rying substitutions at aa 467 and 468 and at aa 477 gave a pattern of binding to mutant oligonucleotide probes that implicates these particu lar amino acids in DNA recognition. EBNA-1 appears to utilize novel me chanisms for both DNA recognition and dimerization since neither domai n conforms to previously described structural motifs.