Figure 4

UV-DDB binds to the nucleosomal apyrimidinic DNA with a different translational position.

(a) Schematic representations of reconstituted nucleosomes containing single AP(outside) and single AP(outside+21). The original AP(outside) position and the AP(outside+21) position are represented by red and blue stars, respectively. The arrow indicates the nucleosomal dyad. (b) Gel electrophoretic mobility shift assay for UV-DDB binding to single AP nucleosomes. Nucleosome core particles (NCP; 5 nM) containing undamaged DNA (lanes 1–6), AP(outside) (lanes 7–12), single AP(outside) (lanes 13–18) and AP(outside+21) (lanes 19—24) were incubated with UV-DDB. The UV-DDB concentrations are 0 nM (lanes 1, 7 and 13), 2.5 nM (lanes 2, 8, and 14), 5 nM (lanes 3, 9 and 15), 10 nM (lanes 4, 10, and 16), 20 nM (lanes 5, 11 and 17) and 40 nM (lanes 6, 12 and 18). (c) Time course experiments. Nucleosome core particles (NCP; 5 nM) containing undamaged DNA (lanes 1–6), single AP(outside) (lanes 7–12) and single AP(outside+21) (lanes 13–18) were incubated with UV-DDB (10 nM) for 1 min (lanes 2, 8 and 14), 3 min (lanes 3, 9 and 15), 5 min (lanes 4, 10, and 16), 10 min (lanes 5, 11 and 17) and 30 min (lanes 6, 12 and 18). Lanes 1, 7 and 13 are the experiments without UV-DDB. (d) Graphic representation of the time course experiments shown in panel (c). Standard deviation values are shown (n = 3).