A number of studies have demonstrated that the herpes simplex virus type 1 (HSV-1) UL9 protein, which is a homodimer in solution, binds to two high affinity binding sites in each origin of replication. Interaction between the proteins bound at the two sites leads to the formation of a complex nucleoprotein structure. The simplest models for this binding interaction predict two possible binding stoichiometries: 1) one UL9 dimer is bound at each site; or 2) one UL9 monomer is bound at each site so that one UL9 dimer occupies both sites. Two recent papers have addressed this issue by using indirect methods to measure the binding stoichiometry. Martin et al. (Martin, D. W., Muñoz, R. M., Oliver, D., Subler, M. A., and Deb, S.(1994) Virology 198, 71-80) reported that a monomer of UL9 binds to a single high affinity site, and Stabell and Olivo (Stabell, E. C., and Olivo, P. D.(1993) Nucleic Acids Res. 21, 5203-5211) concluded that a dimer of UL9 binds to a single high affinity site. We have directly measured the stoichiometry of binding of the carboxyl-terminal DNA binding domain of UL9 (t-UL9) to the origin of replication using a double-label gel shift assay. Using a short synthetic double-stranded oligonucleotide containing a single UL9 binding site, one protein-DNA complex was detected in the gel shift assay, and the molar ratio of UL9 DNA binding domains to DNA binding sites in this complex was determined to be 2.0 ± 0.1 (n = 13). Using the minimal origin sequence excised from plasmid DNA, two protein-DNA complexes were detected. The binding stoichiometry of the faster migrating complex was 1.8 ± 0.1 (n = 15), and the stoichiometry of the more slowly migrating band was 3.7 ± 0.4 (n = 15). The simplest explanation for these data is that UL9 binds to the origin of replication as a homodimer with one dimer bound at both high affinity sites.

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