In:
Proceedings of the National Academy of Sciences, Proceedings of the National Academy of Sciences, Vol. 110, No. 8 ( 2013-02-19)
Kurzfassung:
Our results reveal the essential roles of motor protein regulatory domains in the context of transcription regulation and maintenance of genome integrity and provide a mechanistic explanation for the transcription defects observed in patients with TTD that involve a noncatalytic domain. The XPD helicase is not required for transcription, and the limited capacity of the TFIIH-containing mutant XPD-C259Y to stimulate transcription/transactivation results from the perturbed interaction of XPD with CAK. We suggest that this mutation leads to a conformational change that impedes positioning of TFIIH and its kinase module within transactivation complexes. This conformational change may explain in vitro data showing that the recombinant mutant TFIIH is unable to phosphorylate the C-terminal domain of RNA Pol II (and thus the transcription defect) as well as the absence of TFIIH recruitment on the RARβ2 promoter in response to all trans -retinoic acid. Together, our findings show that the ARCH domain of XPD not only plays a direct role in catalysis but also constitutes a platform for the recruitment of CAK and thus is essential for transcription initiation and regulation. CAK is released from the core-TFIIH during NER following the arrival of xeroderma pigmentosum group A around the DNA lesion ( 6 ). Our results identify the ARCH domain of XPD as a potential molecular switch that may control TFIIH composition and play a key role in the conversion of TFIIH from a factor active in transcription into a DNA repair factor. Previous work demonstrated the role of the XPD/CAK interaction in regulating both transcription and DNA repair. During the process of transcription, the presence of CAK allows phosphorylation of activators as well as of RNA polymerase II (RNA Pol II) to initiate RNA synthesis. In DNA repair, CAK is released from TFIIH upon the arrival of the other factors. By investigating the consequences of mutations identified in patients with TTD ( 5 ), a rare autosomal recessive multisystem disorder whose diagnostic hallmark is brittle hair and which in many patients is associated with a defect in nucleotide excision repair (NER), we have obtained insights on the XPD helicase and have found that the ARCH domain is critical for the recruitment of the CAK complex. Our findings establish that the ARCH domain of XPD interacts directly with the MAT1 subunit of CAK and that the C259Y mutation, which impairs this interaction, strongly influences the in vitro NER and transcription activities of TFIIH. DNA repair defects of TFIIH harboring the C259Y mutation in XPD result from impaired DNA binding and from the inability of the mutant protein to function as a helicase. The direct involvement of the ARCH domain in DNA recognition is fully supported by mutagenesis and structural data obtained from archeal XPD, suggesting that the translocated DNA strand protrudes through the pore formed by the first helicase motor domain, the iron-sulfur cluster, and the ARCH domain ( 2 ). The basal transcription/DNA repair factor TFIIH is organized in two subcomplexes, a six-subunit core and the trimeric CDK-activating kinase (CAK) ( 1 ). These complexes are bridged by the xeroderma pigmentosum group D (XPD) helicase, which, apart from two canonical helicase domains, is composed of a 4FeS cluster domain involved in DNA damage recognition and a recently described module known as the “ARCH domain” ( 2 – 4 ), whose function remains uncharacterized ( Fig. P1 ). Gene expression is regulated at several levels, one of them being transcription initiation. The initiation of RNA synthesis requires several factors, such as the transcription factor IIH (TFIIH). Besides its role in transcription, TFIIH plays a crucial role in maintaining genome integrity through its function in DNA repair. Moreover, mutations in some of the TFIIH subunits result in genetic disorders such as xeroderma pigmentosum, Cockayne syndrome, and trichothiodystrophy (TTD). Therefore it is important to understand the structure and function of this multicomplex to improve our understanding of DNA repair and transcription defects and of the clinical features of these patients.
Materialart:
Online-Ressource
ISSN:
0027-8424
,
1091-6490
DOI:
10.1073/pnas.1213981110
Sprache:
Englisch
Verlag:
Proceedings of the National Academy of Sciences
Publikationsdatum:
2013
ZDB Id:
209104-5
ZDB Id:
1461794-8
SSG:
11
SSG:
12
