A 3′→ 5′ XPB Helicase Defect in Repair/Transcription Factor TFIIH of Xeroderma Pigmentosum Group B Affects Both DNA Repair and Transcription

Jae Ryoung Hwang,Vincent Moncollin,Wim Vermeulen,Thierry Seroz,Hanneke Van Vuuren,Jan H.J Hoeijmakers,Jean Marc Egly

doi:10.1074/jbc.271.27.15898

Abstract

XPB is a subunit of the basal transcription factor TFIIH, which is also involved in nucleotide excision repair (NER) and potentially in cell cycle regulation. A frameshift mutation in the 3'-end of the XPB gene is responsible for a concurrence of two disorders: xeroderma pigmentosum (XP) and Cockayne's syndrome (CS). We have isolated TFIIH from cells derived from a patient (XP11BE) who carries this frameshift mutation (TFIIHmut) and from the mother of this patient (TFIIHwt) to determine the biochemical consequences of the mutation. Although identical in composition and stoichiometry to TFIIHwt, TFIIHmut shows a reduced 3' --> 5' XPB helicase activity. A decrease in helicase and DNA-dependent ATPase activities was also observed with the mutated recombinant XPB protein. The XPB mutation causes a severe NER defect. In addition, we provide evidence for a decrease in basal transcription activity in vitro. The latter defect may provide an explanation for many of the XP and CS symptoms that are difficult to rationalize based solely on an NER defect. Thus, this work presents the first detailed analysis of a naturally occurring mutation in a basal transcription factor and supports the concept that the combined XP/CS clinical entity is actually the result of a combined transcription/repair deficiency.

Highlights

TFIIH, previously called BTF2, was originally defined as a mammalian transcription factor required for accurate transcription of protein-coding genes
Using a monoclonal antibody directed against the recombinant XPB protein, we showed that this polypeptide is present in both cell lines and possesses the predicted molecular weight (Fig. 1C)
An antibody raised against an oligopeptide corresponding to the frameshifted carboxyl terminus of the mutated XPB polypeptide of XP11BE (Ab-CTmut) (Fig. 1B) revealed the presence of the mutant XPB gene product in the TFIIH fraction derived from the patient’s cell line (TFIIHmut) (Fig. 1C, lane 9)

Summary

Introduction

TFIIH, previously called BTF2, was originally defined as a mammalian transcription factor required for accurate transcription of protein-coding genes. Case, a C to A transversion in the last intron of the XPB gene generates a splice mutation at the RNA level [23] and a frameshift at the protein level changing the last 41 amino acids (see Fig. 1B) This patient had acute sun sensitivity with the pigmentation abnormalities characteristic of XP, including actinic keratoses and several cutaneous malignancies, in addition to all the clinical hallmarks of CS such as developmental and neurological abnormalities (dwarfism, microencephaly, and deafness) and impaired sexual development [24, 25]. Our results show that the mutated XPB polypeptide associated with TFIIH presents both helicase and ATPase deficiencies that directly or indirectly impair the NER reaction and, to a lesser extent, appear to affect basal transcription These results provide an explanation for the large range of clinical symptoms associated with CS and TTD, some of which are unrelated to an NER deficiency

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Conclusion