Figure 4.

Mutations in R-E-D and ThM motifs impair the ATPase activity of XPB. (A) A measure of 25 and 75 ng of TFIIH(WT), TFIIH/XPB(E253A), XPB(E253A/R283A), XPB(K346R), XPB(E473A) or XPB(Δ516–526) was tested in a dual incision assay (NER, upper panel) or in a reconstituted transcription assay (Tx, lower panel) as described (Coin et al, 2004). Lane 1 contains highly purified Hela TFIIH (Giglia-Mari et al, 2004). Lane 2 contains all the factors except TFIIH. The sizes of the incision or transcription products are indicated. The transcription and repair signals were quantified using Genetool (Syngene). (B) A measure of 100 ng of the various TFIIH complexes were tested in a KMnO₄ footprint assay (see Figure 1B). Lane 1; Pt-DNA with BSA only. Residues are numbered with the central thymine of the crosslinked GTG sequence designated T0. Arrows indicate KMnO₄ sensitive sites. Adducted strand residues to the 3′ and 5′ of T0 are denoted by positive and negative integers (+N, –N).