Figure 4

Superhelicity relaxation induced nucleosome destabilization. (A and B) Comparison of the changes of superhelicity (A) and nucleosome destabilization (B) in the case of the intercalator SYBR Gold, in Jurkat cells. (A) Determination of the relaxation concentration of SYBR Gold. The average halo radius of G1 phase cells was measured at increasing dye concentrations and in the presence of different salt concentrations (see “winding assay” in Methods). The inset shows the principle of the nuclear halo winding assay: As the intercalator concentration increases, the negatively supercoiled DNA loops get relaxed (halo size is increased), then overwound (i.e. becoming positively supercoiled with a decreased halo size). (B) Histone elution using SYBR Gold at 0.75 M NaCl. The SYBR Gold concentration inducing complete halo relaxation at 0.75 M NaCl concentration and where the DNA loops are completely relaxed is shown by the dashed line on both panels. (See also: Supplementary Fig. S8A) The concentration of the intercalators are shown in a logarithmic scale. Normalization was as described in the Methods. (C and D) Effect of nickase (C) and DNase I (D) treatment on H2A.X salt elution in HCT116 nuclei. (See also: Supplementary Fig. S8C and D). (E and F) Effect of nickase (E) and DNase I (F) treatment on H2A salt elution in HCT116 nuclei. (See also: Supplementary Fig. S8E and F). The elution curves refer to G1 phase cells gated according to their DNA fluorescence intensity distribution. Error bars represent SEM of ~600 G1 nuclei measured by LSC.