FIG. 5.

Evidence for the formation of Rep and TBP complexes on the minimal p5D10 promoter element. (A) Enhanced binding of Rep68 to the p5D10 element in the presence of human TBP. EMSA of radiolabeled p5D10 (lanes 1 to 4) or p5D12 (lanes 5 to 8) DNA fragments incubated with either purified human TBP, Rep68, or both proteins. (B) Competition EMSA. The binding was performed using radiolabeled p5D10 DNA substrate incubated either alone or with the indicated proteins. The binding reactions were then completed with an excess (0-, 30-, 100-, or 300-fold) of unlabeled p5D10 competitor DNA and kept at 30°C for an additional 30 min. (C) Quantification of the amount of bound p5D10 labeled DNA. The amount of free DNA (F) in each lane was quantified by PhosphorImager analysis of the gel presented in panel B and the % of bound p5D10 DNA was calculated by subtracting each value from that obtained in the absence of any protein (lane 1). The asterisk in panels A and B indicates the position of the wells in the gel.