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. 2007 Oct 18;26(22):4768–4776. doi: 10.1038/sj.emboj.7601894

Figure 4.

Figure 4

The XPA67−80 peptide is an effective inhibitor of NER activity. (A) XPA67−80 inhibits the in vitro NER reaction, whereas the mutant XPA67−80 F75A peptide has no effect. HeLa cell extracts were incubated with a plasmid containing a 1,3-intrastrand cisplatin adduct in the presence of increasing concentrations of either XPA67−80 or XPA67−80 F75A (lane 1, no XPA; lanes 2 and 7, 46 nM XPA peptide; lanes 3 and 8, 460 nM; lanes 4 and 9, 4.6 μM; lanes 5 and 10, 46 μM; lanes 6 and 11, 92 μM). Products were visualized by a fill-in reaction following annealing to an oligonucleotide complementary to the excision product with a 4-nt overhang (Shivji et al, 1999). The marker DNA ladder is labeled LMW DNA ladder. (B) XPA67−80 and XPA67−80 F75A do not affect the intrinsic nuclease activity of ERCC1-XPF. The stem12-loop22 substrate (6.6 nM) was incubated with different concentrations of ERCC1-XPF (lanes 2, 4 and 6: 6.7 nM ERCC1-XPF; lanes 3, 5 and 7: 26.8 nM) and 0.4 mM MnCl2 in the presence of no peptide (lanes 1–3), 92 μM XPA67−80 (lanes 4 and 5), and 92 μM XPA67−80 F75A (lanes 6 and 7). The DNA substrate and the cleavage products are indicated.