Effect of number and position of EBNA-1 binding sites in Epstein-Barr virus oriP on the sites of initiation, barrier formation, and termination of replication.
DNA replication intermediates of three plasmids containing all or part of a modified Epstein-Barr virus cis-acting plasmid maintenance region (oriP) were examined to further investigate oriP function. Replication intermediates were analyzed in vivo and in vitro by neutral-neutral two-dimensional gel electrophoresis. The major functional components of the wild-type oriP are a 140-bp dyad symmetry region (single dyad) and 20 tandem copies of a repeat with a 30-bp consensus sequence (family of repeats). A modified oriP was constructed by replacing the family of repeats with three tandem copies of the single dyad (D. A. Wysokenski and J. L. Yates, J. Virol. 63:2657-2666, 1989). Initiation was observed in vivo near the single dyad in the modified oriP, as seen in the wild-type oriP (T. A. Gahn and C. L. Schildkraut, Cell 58:527-535, 1989), but was not observed near the tandem dyads. A replication barrier and termination were observed near the tandem dyads and were similar to those observed at the family of repeats of the wild-type oriP (Gahn and Schildkraut, Cell 58:527-535, 1989). In vitro experiments indicate that the viral trans-acting factor EBNA-1 contributes to efficient barrier formation at the tandem dyads as observed in the family of repeats of the wild-type oriP (V. Dhar and C. L. Schildkraut, Mol. Cell. Biol. 11:6268-6278, 1991). The tandem dyads thus appear to function in a manner similar to the family of repeats. There are significant structural differences between the family of repeats and tandem dyads. The relationship between the number and relative positions of EBNA-1 binding sites in relation to the functions of the family of repeats and the dyad symmetry element is discussed.
Department of Cell Biology, Albert Einstein College of Medicine, New York, New York 10461.
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Platt
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Tcherepanova
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Schildkraut
D000956:Antigens, Viral / Q000378:metabolism*; D001402:B-Lymphocytes / Q000166:cytology; D002478:Cells, Cultured; D004261:DNA Replication; D004279:DNA, Viral / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D019309:Epstein-Barr Virus Nuclear Antigens; D004854:Herpesvirus 4, Human / Q000235:genetics / Q000254:growth & development*; D010957:Plasmids / Q000235:genetics / Q000378:metabolism*
D000956:Antigens, Viral; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D019309:Epstein-Barr Virus Nuclear Antigens