Binding of EBNA-1 to DNA creates a protease-resistant domain that encompasses the DNA recognition and dimerization functions.
The Epstein-Barr virus nuclear antigen EBNA-1 is essential for replication of the viral DNA during latency. EBNA-1 binds as a dimer to palindromic recognition sequences within the plasmid origin of replication, ori-P. In this study, proteinase K susceptibility has been used to further characterize the DNA-binding domain of EBNA-1. Limited protease digestion of EBNA-1 (amino acids 408 to 641) generated a smaller DNA-binding species that had a degree of inherent protease resistance. When EBNA-1 was preincubated with a specific DNA probe, the protease resistance of the smaller binding species increased 100-fold, suggesting that the conformation of EBNA-1 changes on binding. The protease-resistant species comprised an 18-kDa polypeptide that was further cleaved at high levels of protease to 11- and 5.4-kDa products. A model of the proposed protease-resistant domain structure is presented. Constructions carrying serial, internal deletions across the 18-kDa domain were created. Each of the deletions perturbed dimerization ability and abolished DNA binding. These studies suggest that the DNA-binding and dimerization motifs of EBNA-1 lie within a conformationally discrete domain whose overall integrity is necessary for EBNA-1-DNA interaction.
Shah
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Department of Pharmacology and Molecular Sciences, Johns Hopkins School of Medicine, Baltimore, Maryland 21205.
Ambinder
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Hayward
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Hayward
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D000956:Antigens, Viral / Q000187:drug effects / Q000378:metabolism*; D004252:DNA Mutational Analysis; D004279:DNA, Viral / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000187:drug effects / Q000378:metabolism*; D019286:Endopeptidase K; D019309:Epstein-Barr Virus Nuclear Antigens; D004854:Herpesvirus 4, Human / Q000187:drug effects / Q000378:metabolism*; D008954:Models, Biological; D009690:Nucleic Acid Conformation; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics*; D012697:Serine Endopeptidases / Q000494:pharmacology
D000956:Antigens, Viral; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D019309:Epstein-Barr Virus Nuclear Antigens; D012697:Serine Endopeptidases; D019286:Endopeptidase K