Fig. 1

DNA damage at active transcription sites induces reversible transcription inhibition by the DART system. (A) Scheme of the DART system for inflicting genome locus-specific damage. KR generates ROS upon visible light exposure and induces localized damage at transcription either off or on sites. (B and C) TA-KR transfected U2OS TRE cells were irradiated with light for 10 min followed by the indicated postlight incubation time. Phosphorylated POLII detected by anti-4H8 (B) or YFP-POLII (C) is shown at the indicated time points. Quantification of the average fold increase of 4H8 foci intensity of 10 cells at TA-KR damage sites is shown, and error bars indicate the SEM of three independent experiments. Dephosphorylation of POLII but not the dissociation of POLII was observed upon damage. (D) The percentage of CKL-CFP positive cells (defined as CFP foci > 10/cell) in tetR-KR or TA-cherry/KR transfected U2OS TRE cells before and after damage induction is shown. n = 50, and error bars indicate the SEM of three independent experiments.