Figure 3.
S751E mutation inhibits the 5′ incision. (A) Schematic diagram of assays used to detect dual and single incisions. (B) Left panel: Ab-hXPB immunoprecipitated TFIIH-wt (lanes 3 and 4), TFIIH-fs740 (lanes 5 and 6), TFIIH-S751A (lanes 7 and 8) and TFIIH-S751E (lanes 9 and 10) from 100 and 200 μg of 27-1 cell extracts were tested for their ability to promote either the dual (upper panel), 3′ (middle) or 5′ (lower) incisions. Right panel: A measure of 50 and 100 ng of either TFIIH-wt or TFIIH-fs740, immunopurified from GM1855 or GM2252 (XP11BE) cell extracts respectively, was similarly tested. Lane C contains all the NER factors except TFIIH. (C) A 200 ng portion of immunoprecipitated rXPB expressed in baculovirus-infected insect cells was tested either in helicase assay using a bidirectional probe (Coin et al, 1998) or resolved by SDS–PAGE followed by Western blotting (WB). XPB-GKT contains a mutation in the ATPase domain (Guzder et al, 1994). Δ: probe has been heated for 5 min at 100°C. C: control without XPB.