Fig. 3

The recruitment of HR factors at TA-KR damage sites is dependent on active transcription and CSB. (A) Colocalization of RAD51C or RAD52 (green) and TA-KR (red) in cdt1 (blue) expressing U2OS-TRE cells 30 min after KR activation. (B) Quantification of the percentage of cells with positive foci at TA-KR (n = 100) in double thymidine synchronized G1 cells. Error bars indicates the SEM of three independent experiments, and the P values were determined by using Student’s two-tailed t test. (C and D) α-amanitin and DRB treatment abolish the recruitment of RAD51 and RAD51C at TA-KR. RAD52/RAD51C and TA-KR transfected U2OS TRE cells were pretreated with the polymerase II inhibitor (POLIIi) α-amanitin (100 µg/mL) for 1 h or DRB (20 µM) for 24 h or KU55933 (10 µM) for 1 h, then irradiated with light for 10 min followed by incubation for 30 min. Images and quantification of the fold increase of RAD52/RAD51C foci intensity at TA-KR damage sites are shown. (E) The fold increase of RAD52/RAD51C foci intensity of 10 cells at TA-KR damage sites is shown after siRNA treatment of the indicated protein. Data are represented as mean ± SEM, and the P values were determined by using Student’s two-tailed t test. (F) RNaseH (15 units) treatment for 15 min abolishes EU incorporation and the recruitment of RAD52 at TA-KR after damage induction. Images with light irradiation for 10 min followed by 30 min incubation after RNaseH treatment are shown. (G) The percentage of cells with RAD52 or RAD51C foci of 100 cells at TA-KR damage sites is shown after RNaseH treatment. Error bars indicate the SEM of three independent experiments.