Fig. 3. SAP130 and TFTC preferentially bind to UV-damaged DNA. A ³²P-labelled DNA fragment was UV irradiated at different doses as indicated, incubated with increasing amounts of either immunopurified SAP130 (A) or TFTC (B) and tested for retention on nitrocellulose filters. The amounts (µl) of SAP130 and TFTC used in this assay were previously normalized in Figure 1A. Graphs represent the amount of labelled DNA retained on the filters in arbitrary units (AU). AUs were obtained after phosphoimager scanning of the nitrocellulose filters and by deducting the buffer-only background from every TFTC- or SAP130-containing sample. Similar and comparable results (± 5%) were obtained in at least three independent experiments. (C) UV-irradiated DNA inhibits TFTC-mediated Pol II transcription in vitro. TFTC and TFIIDβ were pre-incubated for 10 min with 25 ng of templates, containing the AdMLP or the β-globin (Glob) promoter, in the absence or presence (100 ng) of either UV-irradiated (compet. +UV) or non-UV-irradiated (compet. –UV) competitor DNA fragments. Then the heat-treated HeLa whole-cell extract (WCE, 45°C) was added for 20 min and transcription initiated. The positions of the correctly initiated transcripts from the AdMLP (+1) and the Glob (+1) promoters, determined by quantitative S1 mapping, are indicated. (D) Nucleosomal arrays were assembled as in Figure 5A and the binding of TFTC (10 µl) to the non-irradiated (–) or UV-C-irradiated (2.5 J/cm²) templates was analysed by western blotting with the indicated antibodies. (E) Preferential recruitment of TFTC components and the repair damage recognition protein XP-A from a repair-competent HeLa NE onto bead-linked UV-damaged DNA (2.5 J/cm²) (UV-C), but not bead-linked DNA containing single-stranded breaks (DNase I). The different bead-linked DNA fragments were incubated with the repair-competent HeLa NE (50 µg), washed, and bound proteins were analysed by western blotting. Chromatin assembly factor-1 (CAF-1 p60) is recruited during the repair of both types of damage.