DNA binding activity is required for EBNA 1-dependent transcriptional activation and DNA replication.
Epstein-Barr virus nuclear antigen 1 (EBNA 1) has been shown to be a sequence-specific DNA binding protein that is required for the replication of episomal elements carrying the viral origin of DNA replication, oriP, as well as for the activation of a specific transcriptional enhancer. We have constructed and analyzed a series of deletion and nonsense mutants in a cloned copy of the EBNA 1 gene and have tested mutant peptides for the ability (a) to bind to a synthetic oligonucleotide containing a consensus EBNA 1 binding site, (b) to activate the EBNA 1-specific enhancer, and (c) to drive replication of an oriP-bearing plasmid in a transient replication assay. The presence of a DNA binding domain in the carboxy-terminal third of the protein was confirmed. Interestingly, neither the acidic tail nor the Gly-Ala copolymer of EBNA 1 contributes significantly to binding. In addition to sequences in the carboxy-terminal portion of the protein, our data indicate that sequences in the amino-terminal portion of the polypeptide affect the binding of EBNA 1 to its target sequence. Further, we show that EBNA 1 binds to its recognition sequence as a dimer. Results of transient expression assays indicate that the ability of EBNA 1 species to activate the transcriptional enhancer and to drive the replication of oriP plasmids is directly dependent on the ability of the polypeptides to bind to the EBNA 1 consensus binding sequence.
Polvino-Bodnar
M
M
Department of Biology, Northeastern University, Boston, Massachusetts 02115.
Schaffer
P A
PA
D000956:Antigens, Viral / Q000737:chemistry / Q000502:physiology*; D001483:Base Sequence; D003001:Cloning, Molecular; D004252:DNA Mutational Analysis; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000502:physiology*; D004742:Enhancer Elements, Genetic; D019309:Epstein-Barr Virus Nuclear Antigens; D015967:Gene Expression Regulation, Viral; D004854:Herpesvirus 4, Human / Q000235:genetics*; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D010957:Plasmids; D012045:Regulatory Sequences, Nucleic Acid; D013329:Structure-Activity Relationship; D014158:Transcription, Genetic; D015533:Transcriptional Activation
D000956:Antigens, Viral; D004268:DNA-Binding Proteins; D019309:Epstein-Barr Virus Nuclear Antigens; D009838:Oligodeoxyribonucleotides