FIG. 5.

Gal4-VP16 does not block nucleosome sliding by ISW2. Gal4VP16 was prebound to nucleosomes for 30 min at 30°C in reaction mixtures containing 300 μM ATP, 4.6 pmol of Gal4-VP16, and 3.4 pmol of nucleosomes, and remodeling was started by addition of 0.2 pmol of ISW2 for 1 to 15 min (lanes 4 to 13). After remodeling, Gal4-VP16 was competed with 25 pmol of a short DNA fragment containing single Gal4 binding site for 5 min at 30°C (lanes 3 and 10 to 13). To stop remodeling during competition, the reaction mixture was supplemented with 10 mM γ-S-ATP and 3 μg of salmon sperm DNA. The reactions were analyzed by gel shift on 5% polyacrylamide native PAGE (acrylamide/bisacrylamide ratio, 60/1) gel containing 0.2× TBE at 4°C. In lane 10, the salmon sperm DNA and γ-S-ATP were added before the addition of ISW2.