UV-induced DNA lesions trigger ATP-dependent chromatin remodeling. (A–C) Distribution of GFP-tagged histones H1.0 (A), H2A (B), or H4 (C) at sites of local damage (100 J/m2) in MRC5 cells marked by the accumulation of DDB2-mCherry. (D) GFP-H4 and DDB2-mCherry distribution at local UV damage after ATP depletion. (E) Quantification of the intensity of GFP-tagged histones at sites of UV-induced DNA damage shown in a box plot (∼50 cells for each condition). An intensity of 1 represents the mean intensity in the nucleus (excluding nucleoli), and intensities <1 represent a reduced histone density in the locally damaged area. The dotted line represents a ratio of 1, which means that the intensity in the local damage is the same as the intensity elsewhere in the nucleus. Asterisks indicate significant differences based on a t test (P < 0.05). Open circles are data points that are considered outliers, as their value is greater than the upper quartile, +1.5 the interquartile distance, or less than the lower quartile, −1.5 the interquartile distance. (F) Distribution of GFP-H1 and DNA stained by DAPI in U2OS cells after local damage (100 J/m2) through 5-µm pores. Cells were stained for endogenous DDB2. (G) Real-time imaging of DDB2-YFP in living cells after local UV irradiation (100 J/m2 through 5-µm pores at 37°C) revealed the appearance of a fiberlike structure emanating from within the locally damaged area. (H and I) A micrograph (H) and quantitative (I) analysis of the distribution of GFP-H4 pixel intensities (normalized between 0 and 100%) inside the locally UV-damaged area (red circle in H and red bars in the histogram shown in I) or inside a similarly sized nondamaged area in the nucleus (blue circle in H and blue bars in the histogram shown in I). (J and K) Similar analysis for an ATP-depleted cell.
