Separation of the complex DNA binding domain of EBNA-1 into DNA recognition and dimerization subdomains of novel structure

Mei Ru Chen, Jaap M. Middeldorp, S. Diane Hayward

Research output: Contribution to journalArticle

Abstract

EBNA-1 is essential for replication of the latent episomal form of the Epstein-Barr virus genome and is involved in regulation of viral latency 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 created 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 heterodimerize with a wild-type EBNA-1 (aa 459 to 641) polypeptide was tested in immunoprecipitation assays with a monoclonal antibody, EBNA.OT1x, that recognizes EBNA-1 (aa 408 to 641) but not EBNA-1 (aa 459 to 641). These experiments revealed that mutations affecting dimerization occurred over two separate regions, aa 501 to 532 and aa 554 to 598. DNA binding was tested in mobility shift assays against a panel of oligonucleotide-binding sites. Dimerization was a prerequisite for DNA binding. The DNA recognition domain was localized to a separate region, aa 459 to 487, upstream of the dimerization domain. EBNA-1 variants carrying substitutions at aa 467 and 468 and at aa 477 gave a pattern of binding to mutant oligonucleotide probes that implicates these particular amino acids in DNA recognition. EBNA-1 appears to utilize novel mechanisms for both DNA recognition and dimerization since neither domain conforms to previously described structural motifs.

Original languageEnglish (US)
Pages (from-to)4875-4885
Number of pages11
JournalJournal of Virology
Volume67
Issue number8
StatePublished - Aug 1993

Fingerprint

DNA-binding domains
dimerization
Dimerization
Amino Acids
amino acids
DNA
EBV-encoded nuclear antigen 1
Virus Latency
Oligonucleotide Probes
Human herpesvirus 4
Sequence Deletion
mutants
oligonucleotide probes
Electrophoretic Mobility Shift Assay
Amino Acid Substitution
Human Herpesvirus 4
Immunoprecipitation
Point Mutation
assays
Oligonucleotides

ASJC Scopus subject areas

  • Immunology

Cite this

Separation of the complex DNA binding domain of EBNA-1 into DNA recognition and dimerization subdomains of novel structure. / Chen, Mei Ru; Middeldorp, Jaap M.; Hayward, S. Diane.

In: Journal of Virology, Vol. 67, No. 8, 08.1993, p. 4875-4885.

Research output: Contribution to journalArticle

Chen, Mei Ru ; Middeldorp, Jaap M. ; Hayward, S. Diane. / Separation of the complex DNA binding domain of EBNA-1 into DNA recognition and dimerization subdomains of novel structure. In: Journal of Virology. 1993 ; Vol. 67, No. 8. pp. 4875-4885.
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