Abstract
The ten-subunit transcription factor IIH (TFIIH) plays a crucial role in transcription and nucleotide excision repair (NER). Inactivating mutations in the smallest 8-kDa TFB5/TTDA subunit cause the neurodevelopmental progeroid repair syndrome trichothiodystrophy A (TTD-A). Previous studies have shown that TTDA is the only TFIIH subunit that appears not to be essential for NER, transcription, or viability. We studied the consequences of TTDA inactivation by generating a Ttda knock-out (Ttda−/−) mouse-model resembling TTD-A patients. Unexpectedly, Ttda−/− mice were embryonic lethal. However, in contrast to full disruption of all other TFIIH subunits, viability of Ttda−/− cells was not affected. Surprisingly, Ttda−/− cells were completely NER deficient, contrary to the incomplete NER deficiency of TTD-A patient-derived cells. We further showed that TTD-A patient mutations only partially inactivate TTDA function, explaining the relatively mild repair phenotype of TTD-A cells. Moreover, Ttda−/− cells were also highly sensitive to oxidizing agents. These findings reveal an essential role of TTDA for life, nucleotide excision repair, and oxidative DNA damage repair and identify Ttda−/− cells as a unique class of TFIIH mutants.
Highlights
DNA-damaging agents are a constant challenge to DNA integrity
We demonstrate that Ttda2/2 mice are embryonic lethal
We show that Ttda2/2 mouse cells are the first known viable transcription factor II H (TFIIH) subunit knock-out cells, which are completely Nucleotide excision repair (NER) deficient and sensitive to oxidative agents
Summary
A network of DNA-repair systems collectively removes most lesions and safeguards the stability of the genome [1]. Nucleotide excision repair (NER) is one such DNA-repair mechanism capable of removing a wide variety of structurally unrelated DNA helix-distorting lesions, including ultraviolet light (UV)-induced lesions and bulky chemical adducts. DNA repair of helix-distorting lesions requires the helix to be opened at the site of the lesion for efficient incision of the damaged strand [2]. A protein complex essential to this process is basal transcription factor II H (TFIIH). TFIIH was initially identified as a general RNA polymerase II transcription initiation factor [3], this multi-subunit complex was subsequently found to have multiple functions: including RNA polymerase I transcription and, activated transcription and cell cycle control [4,5,6]. TFIIH is composed of two sub-complexes: the 7-subunit core complex comprised of xeroderma pigmentosum group B (XPB), xeroderma pigmentosum group D (XPD), p62, p52, p44, p34 and trichothiodystrophy group A (TTDA), and the associated trimeric CDK-activating kinase (CAK) complex involving CDK7, MAT1 and cyclin H
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