Fig. 2.

DNA repair and helicase activities of rIIHs. (A) In vitro double-incision assay. Increasing amounts of immunopurified rIIH were added to an incision/excision assay using recombinant NER factors, and the reaction was analyzed by electrophoresis followed by autoradiography. “t” corresponds to ∼25 ng of rIIH with p44 as reference. (B) The 5′–3′ helicase activity of XPD variants. Equivalent amounts of each Flag-purified XPD variant (∼100 ng) were added to a 5′-strand extension probe obtained by annealing a 52-nt single-strand DNA to a 5′ ³²P-labeled 25-nt single-stranded DNA in the presence of increasing amounts of p44 (0, 50, or 500 ng). Single- and double-stranded DNA are separated by electrophoresis on a 14% (wt/vol) polyacrylamide gel and analyzed by autoradiography (lanes 3–15). The symbols “−” and “Δ” indicate the native and denatured probes, respectively (lanes 1 and 2). (C) DNA-binding activity. Increasing amounts of XPD variants (Left, lanes 1–9) and CAK-XPD (Right, lanes 10–14) were incubated with the labeled 5′-strand extension probe shown in B, and the resulting nucleoprotein complexes were analyzed by electrophoresis using a 6% (wt/vol) polyacrylamide gel followed by autoradiography with XPD as reference. Densitometric analysis of lanes 3, 5, 7, 9, 12, and 14 are shown as below. “×” corresponds to 100 ng of XPD. BP, bound probe; FB, free probe. (D) 5′–3′ helicase activities of rIIH complexes. Insect cells were infected with a set of baculoviruses overexpressing the subunits of TFIIH including either wild-type or mutated Flag-tagged XPD, and complexes were immunoprecipitated using an antibody directed against the Flag epitope in buffer B. After elution with the Flag synthetic peptide, core-TFIIH/XPD (core-IIH, lanes 1–3) and Holo-TFIIH (rIIH, lanes 4–6) were analyzed by Western blot analysis (WB), and equivalent amounts of complex (∼200 ng) were tested for their 5′–3′ helicase activities (Helicase). The negative control is shown in lane 7. Densitometric analysis suggests a fivefold decrease of Holo-TFIIH (lane 4) 5′–3′ helicase activity compared with that of core-TFIIH (lane 1) in the case of XPD-wt.