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J Biol Chem, Vol. 275, Issue 6, 4258-4266, February 11, 2000

TFIIH with Inactive XPD Helicase Functions in Transcription Initiation but Is Defective in DNA Repair*

G. Sebastiaan Winklerab, Sofia J. Araújocd, Ulrike Fiedleref, Wim Vermeulena, Frederic Coing, Jean-Marc Eglyg, Jan H. J. Hoeijmakersah, Richard D. Woodc, H. Th. Marc Timmerse, and Geert Weedaai

From the a Department of Cell Biology and Genetics, Medical Genetics Center, Erasmus University Rotterdam, P. O. Box 1738, 3000 DR Rotterdam, e Laboratory for Physiological Chemistry, Utrecht University, P. O. Box 80042, 3508 TA Utrecht, The Netherlands, c Imperial Cancer Research Fund, Clare Hall Laboratories, Blanche Lane, South Mimms, Herts, EN6 3LD, United Kingdom, and g Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM, 1 Rue Laurent Fries, B.P. 163, 67404 Illkirch, France

TFIIH is a multisubunit protein complex involved in RNA polymerase II transcription and nucleotide excision repair, which removes a wide variety of DNA lesions including UV-induced photoproducts. Mutations in the DNA-dependent ATPase/helicase subunits of TFIIH, XPB and XPD, are associated with three inherited syndromes as follows: xeroderma pigmentosum with or without Cockayne syndrome and trichothiodystrophy. By using epitope-tagged XPD we purified mammalian TFIIH carrying a wild type or an active-site mutant XPD subunit. Contrary to XPB, XPD helicase activity was dispensable for in vitro transcription, catalytic formation of trinucleotide transcripts, and promoter opening. Moreover, in contrast to XPB, microinjection of mutant XPD cDNA did not interfere with in vivo transcription. These data show directly that XPD activity is not required for transcription. However, during DNA repair, neither 5' nor 3' incisions in defined positions around a DNA adduct were detected in the presence of TFIIH containing inactive XPD, although substantial damage-dependent DNA synthesis was induced by the presence of mutant XPD both in cells and cell extracts. The aberrant damage-dependent DNA synthesis caused by the mutant XPD does not lead to effective repair, consistent with the discrepancy between repair synthesis and survival in cells from a number of XP-D patients.

* This work was supported in part by NWO Grant 901-01-151 from the Netherlands Organization for Scientific Research section on Medical Sciences, Grant EUR-94-763 from the Dutch Cancer Society KWF, and by the Louis Jeantet Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

b Present address: Mechanisms of Transcription Laboratory, Imperial Cancer Research Fund, Clare Hall Laboratories, Blanche Lane, South Mimms, Herts, EN6 3LD, UK.

d Supported by the Portuguese Programa Gulbenkian de Doutoramento em Biologia e Medicina, Fundação para a Ciência e Tecnologia and the Imperial Cancer Research Fund.

f Supported by a long term EMBO fellowship.

h To whom correspondence should be addressed. Tel.: 31-10-4087199; Fax: 31-10-4089468; E-mail: Hoeijmakers@gen.fgg.eur.nl.

i Supported by the Royal Netherlands Academy of Arts and Sciences.

Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
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