Sybr gold

Unless otherwise stated, 0N80 end-positioned nucleosomes with and without DNA lesions (140 nM) were incubated with INO80-C complexes (50 nM) in sliding buffer [25 mM HEPES pH8.0, 50 mM NaCl, 5% glycerol, 1 mM TCEP and 2 mM MgCl₂] in a final volume of 10 μl at room temperature. For reactions presented in Figure 1, different concentrations of NaCl were used. Sliding was initiated by adding 1 mM ATP, and the reaction was quenched by adding 5 mM EDTA and 0.2 mg/ml lambda DNA (NEB). Reactions at different time points were collected and resolved on 6% Native-PAGE gels at 4 °C (100 V, 90 min, 1× TBE). Gels were stained with SYBR-GOLD (GoldBio) before imaging on a Typhoon imager (Cytiva). Quantification of the gels was done using ImageJ software version 1.53e. The percentage of fully remodeled nucleosomes was plotted against time using GraphPad (Prism) software.

To monitor the binding of the enzyme to nucleosomes, 70 nM damaged nucleosomes were incubated with an increased amount of INO80-C ΔN sub-complex in a final volume of 10 μl in the binding buffer [25 mM HEPES pH7.5, 60 mM NaCl, 5% glycerol, 1 mM TCEP]. The reactions were incubated for 10 min at 30 °C before being resolved on 4% Native-PAGE at 4 °C (100 V, 90 min, 1× TBE). The gels were stained with SYBR-GOLD (GoldBio) and imaged by a Typhoon imager (Cytiva).