FIG. 6.

Mechanism of YY1 repression of HPV ori replication. (A) Interaction of YY1 protein with GST-H18E2 protein. A Western blot following SDS–10% PAGE was used to reveal His-YY1 retained on glutathione-beads loaded with increasing amounts of GST-H18E2 fusion protein but not on beads loaded with GST alone. Purified His-YY1 (3 μg, one half of input amount) was loaded in lane 4. A similar result was obtained when the binding assay was performed in the presence of ethidium bromide (30 μg/ml), which eliminates potential nonspecific binding caused by interactions with DNA (24). (B) Partial restoration of HPV-18 ori replication in the presence of exogenous YY1 by elevated E2 protein. Replication reactions of pBS-H18ori was conducted in the absence of the viral replication proteins (lane 1) or in the presence of 180 ng of His-H18E1 plus 14 ng of GST-H18E2 (lanes 2 to 6) or plus 140 ng of E2 (lanes 7 and 8). His-YY1 (lanes 3 and 4) or His-YY1, which was boiled for 10 min under mineral oil (lanes 5 and 6), was added as indicated (in micrograms). The relative intensity (Rel I) values were calculated in percentages by comparing isotope incorporation to that in lane 2, after subtracting the background incorporation in lane 1, as measured by a PhosphorImager.