Figure 5.

RSC and Sth1 displace a triple helix. Triple-helix substrates consisting of a 40-base triple helical region were prepared as described in Materials and Methods. Triple helices were center-positioned on a 190-bp duplex DNA, end-positioned on a 114-bp duplex DNA, or prepared without duplex extension. Substrates were treated as indicated at 30°C for 30 min (or heated briefly at 90°C, labeled Heat) and separated in a 15% polyacrylamide gel. Displacement of the ³²P-labeled third strand as a percentage of total displacement (%Disp.) was quantified by PhosphorImager analysis. (A) Displacement of a center-positioned triple helix by Sth1. (B) Displacement of a center-positioned triple helix by RSC. (C) Sth1 cannot displace an isolated triple helix. Identical results were obtained with 8 nM RSC (data not shown). (D) Displacement of an end-positioned triple helix by Sth1 is not affected by the presence of a nick near the duplex/triplex junction. Intact substrate or an identical substrate bearing a nick 4 bp from the duplex/triplex junction was used. Here, reactions were performed at 30°C for 1 h to compensate for lower efficiency compared with center-positioned substrates. Identical results were obtained with 8 nM RSC (data not shown).