TFIIH is a high molecular weight complex with a remarkable dual function in nucleotide excision repair and initiation of
RNA polymerase II transcription. Mutations in the largest subunits, the XPB and XPD helicases, are associated with three inherited disorders:
xeroderma pigmentosum, Cockayne's syndrome, and
trichothiodystrophy. To facilitate the purification and biochemical characterization of this intricate complex, we generated a cell line stably expressing tagged XPB, allowing the immunopurification of the XPB
protein and associated factors. Addition of two tags, a N-terminal hexameric
histidine stretch and a C-terminal
hemagglutinin epitope, to this highly conserved
protein did not interfere with its functioning in repair and transcription. The
hemagglutinin epitope allowed efficient TFIIH immunopurification to homogeneity from a fractionated whole
cell extract in essentially one step. We conclude that the predominant active form of TFIIH is composed of nine subunits and that there is one molecule of XPB per TFIIH complex. The affinity-purified complex exhibits all expected TFIIH activities:
DNA-dependent ATPase, helicase, C-terminal domain
kinase, and participation in in vitro and in vivo nucleotide excision repair and in vitro transcription. The affinity purification procedure described here is fast and simple, does not require extensive chromatographic procedures, and yields highly purified, active TFIIH.