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 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.
Winkler
G S
GS
Department of Cell Biology and Genetics, Medical Genetics Center, Erasmus University, P. O. Box 1738, 3000 DR Rotterdam, The Netherlands.
Vermeulen
W
W
Coin
F
F
Egly
J M
JM
Hoeijmakers
J H
JH
Weeda
G
G
D015153:Blotting, Western; D002461:Cell Line, Transformed; D002846:Chromatography, Affinity / Q000379:methods*; D004265:DNA Helicases; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000378:metabolism*; D000939:Epitopes / Q000378:metabolism*; D006801:Humans; D051758:Transcription Factor TFIIH; D014157:Transcription Factors / Q000302:isolation & purification*; D019473:Transcription Factors, TFII
D004268:DNA-Binding Proteins; D000939:Epitopes; D014157:Transcription Factors; D019473:Transcription Factors, TFII; C072022:XPBC-ERCC-3 protein; D051758:Transcription Factor TFIIH; D004265:DNA Helicases