|A small molecule screen identifies an inhibitor of DNA repair inducing the degradation of TFIIH and the chemosensitization of tumor cells to platinum. |
Alekseev, S; Ayadi, M; Brino, L; Egly, JM; Larsen, AK; Coin, F
Chemistry & biology
Nucleotide excision repair (NER) removes DNA lesions resulting from exposure to UV irradiation or chemical agents such as platinum-based drugs used as anticancer molecules. Pharmacological inhibition of NER is expected to enhance chemosensitivity but nontoxic NER inhibitors are rare. Using a drug repositioning approach, we identify spironolactone (SP), an antagonist of aldosterone, as a potent NER inhibitor. We found that SP promotes a rapid and reversible degradation of XPB, a subunit of transcription/repair factor TFIIH. Such degradation depends both on ubiquitin-activating enzyme and on the 26S proteasome. Supplementation of extracts from SP-treated cells with purified TFIIH restored TFIIH-dependent repair and transcription activities in vitro, demonstrating the specific impact of SP on two fundamental functions of TFIIH. Finally, SP potentiated the cytotoxicity of platinum derivatives toward tumor cells, making it a potential therapeutic and research tool.
|Sequential and ordered assembly of a large DNA repair complex on undamaged chromatin. |
Ziani, S; Nagy, Z; Alekseev, S; Soutoglou, E; Egly, JM; Coin, F
The Journal of cell biology
In nucleotide excision repair (NER), damage recognition by XPC-hHR23b is described as a critical step in the formation of the preincision complex (PInC) further composed of TFIIH, XPA, RPA, XPG, and ERCC1-XPF. To obtain new molecular insights into the assembly of the PInC, we analyzed its formation independently of DNA damage by using the lactose operator/repressor reporter system. We observed a sequential and ordered self-assembly of the PInC operating upon immobilization of individual NER factors on undamaged chromatin and mimicking that functioning on a bona fide NER substrate. We also revealed that the recruitment of the TFIIH subunit TTDA, involved in trichothiodystrophy group A disorder (TTD-A), was key in the completion of the PInC. TTDA recruits XPA through its first 15 amino acids, depleted in some TTD-A patients. More generally, these results show that proteins forming large nuclear complexes can be recruited sequentially on chromatin in the absence of their natural DNA target and with no reciprocity in their recruitment.
|An Xpb mouse model for combined xeroderma pigmentosum and cockayne syndrome reveals progeroid features upon further attenuation of DNA repair. |
Andressoo, JO; Weeda, G; de Wit, J; Mitchell, JR; Beems, RB; van Steeg, H; van der Horst, GT; Hoeijmakers, JH
Molecular and cellular biology
Patients carrying mutations in the XPB helicase subunit of the basal transcription and nucleotide excision repair (NER) factor TFIIH display the combined cancer and developmental-progeroid disorder xeroderma pigmentosum/Cockayne syndrome (XPCS). Due to the dual transcription repair role of XPB and the absence of animal models, the underlying molecular mechanisms of XPB(XPCS) are largely uncharacterized. Here we show that severe alterations in Xpb cause embryonic lethality and that knock-in mice closely mimicking an XPCS patient-derived XPB mutation recapitulate the UV sensitivity typical for XP but fail to show overt CS features unless the DNA repair capacity is further challenged by crossings to the NER-deficient Xpa background. Interestingly, the Xpb(XPCS) Xpa double mutants display a remarkable interanimal variance, which points to stochastic DNA damage accumulation as an important determinant of clinical diversity in NER syndromes. Furthermore, mice carrying the Xpb(XPCS) mutation together with a point mutation in the second TFIIH helicase Xpd are healthy at birth but display neonatal lethality, indicating that transcription efficiency is sufficient to permit embryonal development even when both TFIIH helicases are crippled. The double-mutant cells exhibit sensitivity to oxidative stress, suggesting a role for endogenous DNA damage in the onset of XPB-associated CS.
|Dynamic interaction of TTDA with TFIIH is stabilized by nucleotide excision repair in living cells. |
Giglia-Mari, G; Miquel, C; Theil, AF; Mari, PO; Hoogstraten, D; Ng, JM; Dinant, C; Hoeijmakers, JH; Vermeulen, W
Transcription/repair factor IIH (TFIIH) is essential for RNA polymerase II transcription and nucleotide excision repair (NER). This multi-subunit complex consists of ten polypeptides, including the recently identified small 8-kDa trichothiodystrophy group A (TTDA)/ hTFB5 protein. Patients belonging to the rare neurodevelopmental repair syndrome TTD-A carry inactivating mutations in the TTDA/hTFB5 gene. One of these mutations completely inactivates the protein, whereas other TFIIH genes only tolerate point mutations that do not compromise the essential role in transcription. Nevertheless, the severe NER-deficiency in TTD-A suggests that the TTDA protein is critical for repair. Using a fluorescently tagged and biologically active version of TTDA, we have investigated the involvement of TTDA in repair and transcription in living cells. Under non-challenging conditions, TTDA is present in two distinct kinetic pools: one bound to TFIIH, and a free fraction that shuttles between the cytoplasm and nucleus. After induction of NER-specific DNA lesions, the equilibrium between these two pools dramatically shifts towards a more stable association of TTDA to TFIIH. Modulating transcriptional activity in cells did not induce a similar shift in this equilibrium. Surprisingly, DNA conformations that only provoke an abortive-type of NER reaction do not result into a more stable incorporation of TTDA into TFIIH. These findings identify TTDA as the first TFIIH subunit with a primarily NER-dedicated role in vivo and indicate that its interaction with TFIIH reflects productive NER.