Affinity purification of human DNA repair/transcription factor TFIIH using epitope-tagged xeroderma pigmentosum B protein.

The Journal of Biological Chemistry
G S WinklerG Weeda

Abstract

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 i...Continue Reading

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Citations

Jan 17, 2004·Biochimie·Renata M A CostaCarlos F M Menck
Oct 20, 1999·Mutation Research·S J Araújo, R D Wood
Sep 5, 2001·Genes to Cells : Devoted to Molecular & Cellular Mechanisms·A FukudaK Hisatake
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Nov 10, 2009·DNA Repair·Denis E KainovArnaud Poterzsman
Jun 24, 2008·Journal of the American Academy of Dermatology·Amy S Cheng, Susan J Bayliss
Oct 30, 2007·Protein Science : a Publication of the Protein Society·Andrew J McCluskeyJean Gariépy
Mar 1, 2005·Journal of Biochemical and Biophysical Methods·Enrique CastañoLuis Carlos Rodriguez Zapata
Nov 28, 2002·Molecular Cell·Deborah HoogstratenAdriaan B Houtsmuller
Oct 17, 1998·The Journal of Biological Chemistry·X B QiuA Dutta
Apr 24, 2001·Environmental and Molecular Mutagenesis·A Ronen, B W Glickman
Jul 25, 2006·Critical Reviews in Biochemistry and Molecular Biology·Mary C Thomas, Cheng-Ming Chiang
Feb 8, 2000·The Journal of Biological Chemistry·G S WinklerG Weeda
Dec 26, 2001·The Journal of Biological Chemistry·Federica Marini, Richard D Wood

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