AFFINITY PURIFICATION OF HUMAN DNA-REPAIR TRANSCRIPTION FACTOR TFIIH USING EPITOPE-TAGGED XERODERMA-PIGMENTOSUM B-PROTEIN

TFIIH is a high molecular weight complex with a remarkable dual functi on in nucleotide excision repair and initiation of RNA polymerase II t ranscription. Mutations in the largest subunits, the XPB and XPD helic ases, are associated with three inherited disorders: xeroderma pigment osum, Cockayne's syndrome, and trichothiodystrophy. To facilitate the purification and biochemical characterization of this intricate comple x, we generated a cell line stably expressing tagged XPB, allowing the immunopurification of the XPB protein and associated factors. Additio n of two tags, a N-terminal hexameric histidine stretch and a C-termin al hemagglutinin epitope, to this highly conserved protein did not int erfere with its functioning in repair and transcription. The hemagglut inin epitope allowed efficient TFIIH immunopurification to homogeneity from a fractionated whole cell extract in essentially one step. We co nclude that the predominant active form of TFIIH is composed of nine s ubunits and that there is one molecule of XPB per TFIIH complex. The a ffinity-purified complex exhibits all expected TFIIH activities: DNA-d ependent ATPase, helicase, C-terminal domain kinase, and participation in in vitro and in vivo nucleotide excision repair and in vitro trans cription. The affinity purification procedure described here is fast a nd simple, does not require extensive chromatographic procedures, and yields highly purified, active TFIIH.