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. Author manuscript; available in PMC: 2010 Jul 10.
Published in final edited form as: Mol Cell. 2009 Jul 10;35(1):58–69. doi: 10.1016/j.molcel.2009.05.013

Figure 1. Template commitment of ISW2 to nucleosomes requires ATP.

Figure 1

(A) Template commitment of ISW2 was examined with equimolar amounts of ISW2 and 70N0 nucleosomes (42 nM), ATP (800 µM), and excess competitor DNA added at the indicated times and incubated at 20°C. Samples were removed at the respective times (circled numbers) and analyzed by gel shift assay as shown on the right. In group IV ISW2 and nucleosomes were incubated together for 10 min before starting the initial start time point. Lane numbers correspond to the time points sampled and lane 1 is nucleosomes without ATP or ISW2. As nucleosomes change their translational position on DNA their electrophoretic mobility shifts from its original position (N1) to progressively more central positions (N2 and N3). (B) Template commitment assays with radioactive (r70N0) and non-radioactive (c70N0) nucleosomes were added at different reaction times to show template-committed complexes were resistant to competition by other nucleosomes. Nucleosome remodeling was initiated by incubating with 28 nM of either radiolabeled 70N0 (r70N0) or non-radiolabeled 70N0 (c70N0) and 28 nM of ISW2 at 20°C in the presence of 90 µM ATP. ISW2 at this molar concentration is completely bound with nucleosomes. Numbers above each time point indicate sample number and corresponding lane number. After each time point, the reaction was stopped and separated by 5% native gel electrophoresis.