Fig. 3. Effects of chromatin structure on NER. (A) Schematic representation of the predicted cleavage products after incision of the two internally labeled 6-4PP substrates, center-dinucleosome DNA (a) and linker-dinucleosome DNA (b). The labeled products of the excision assay are represented by thin bidirectional arrows along with their lengths in nucleotides (nt). Thick arrows indicate the location and orientation of the 120 bp 5S RNA gene. The 5′-internal promoter of two repeats of the 5S RNA gene was mutated by base substitution, and is indicated by the white box in plasmid pXSP5S. Internally radiolabeled sites of center- and linker-dinucleosome DNA are indicated by black and white asterisks, respectively. The major nucleosome core positions are shown by ovals. (B) Excision activity of NER factors on synthetic chromatin templates. Chromatin templates were assembled on internally labeled DNA fragments containing either no 6-4PP (lane 1), 6-4PPs at the nucleosome centers (lanes 2–4) or a 6-4PP in the linker DNA (lanes 5–7). These were used as substrates in the excision assay. Naked DNA (lanes 1, 2 and 5), mononucleosomes (lanes 3 and 6) or dinucleosomes (lanes 4 and 7) were incubated with purified NER factors for 30 min at 30°C. Cleavage products were analyzed by 12% denaturing PAGE. Each value obtained from the excision products is expressed as a percentage of the total signal in each lane, which is shown at the bottom of the lane. This figure is representative of data obtained from repeated trials of independently conducted experiments. (C) The quantitative data from (B) shown as a bar graph. The relative excision activity of dinucleosomes containing 6-4PP at the center of nucleosomes (black bars) or in linker DNA (white bars) was calculated by taking the value of excision on each naked DNA template as 100%.