INO80-Mediated Nucleosome Sliding Dynamics

Nucleosome preparation and sliding by INO80 was performed under conditions as reported earlier^{19 (link)}. Sliding for in the presence of various [INO80] as reported in Fig. 2f (for 1 minute) and Extended Data Fig. 7 (for 1 minute and 2.5 minutes) was performed in 10 μl reaction volumes containing 8 nM nucleosomes (nucleosomes formed on both constructs in the pair were present in equimolar proportion), 2 mM ATP, 24 mM tris-HCl pH 7.5, 43 mM KCl, 2.86 mM MgCl₂, 0.55% glycerol and indicated concentration of INO80. The mixture was incubated without ATP at 30 °C for 7 minutes. After addition of ATP, the reaction was allowed to proceed for 1 minute at 30 °C, and was then quenched by the addition of lambda DNA and ADP to final concentrations of 66.7 μg/ml and 20 mM respectively. For all sliding experiments reported in Extended Data Fig. 6b (timecourse of INO80 sliding), conditions were the same except incubations prior to ATP addition and the subsequent sliding reaction were carried out at room temperature. The reaction was continued for the indicated amounts of time in presence of saturating INO80 prior to quenching. Quenched reactions were loaded on to 6% TBE gels (Invitrogen) in presence of 10% glycerol and run at 150 V for 1.5 hrs. The gel was imaged separately for Cy3 and Cy5 fluorescence.