Chz1 chaperone property activity analysis

The chaperone assay was modified from what was described in the early study [12]. As a control, the 147 bp Widom 601 sequence DNA (0.5 μM) was mixed with scAB or scZB at concentrations of 2, 4, or 8 μM and was subsequently incubated in 20 mM MES (pH 6.0) and 0.5 M NaCl in a total volume of 20 μL. To investigate the effects of Chz1-MC or its mutants, histones (8 μM) were incubated with 0.5, 1, 2, 3, 4, 5, 6, 7, or 8 μM of Chz1-MC or its mutants in 20 mM MES (pH 6.0) and 0.5 M NaCl. Binding of Chz1-MC or its mutants to histones was allowed to proceed at 4°C for 15 min before the addition of DNA. In addition, a DNA-free control containing only Chz1-MC at the concentration corresponding to the highest titration point with DNA alone was also used. Histone mutants were performed using the same protocol; 60% (v/v) sucrose was added to the reaction system after the reactions, precipitates were removed by centrifugation, and the remaining soluble complexes were separated with native PAGE.