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.
Platt
T H
TH
Department of Cell Biology, Albert Einstein College of Medicine, New York, New York 10461.
Tcherepanova
I Y
IY
Schildkraut
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CL
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