Dynamic Interaction of TTDA with TFIIH Is Stabilized by Nucleotide Excision Repair in Living Cells

Giuseppina Giglia-Mari,Catherine Miquel,Deborah Hoogstraten,Wim Vermeulen,Arjan F Theil,Jan H J Hoeijmakers,Pierre-Olivier Mari,Jessica M Y Ng,Christoffel Dinant,Priscilla Cooper

doi:10.1371/journal.pbio.0040156

Abstract

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.

Highlights

DNA-damaging agents constantly challenge the integrity of DNA
Within the cytoplasm (Figure 3A), the mobility curve of trichothiodystrophy group A (TTDA)-green fluorescent protein (GFP) virtually overlaps with that of non-tagged GFP and both show a significant faster fluorescence recovery than cytoplasmic xeroderma pigmentosum complementation group D (XPD)-GFP. These results suggest that the majority of TTDA-GFP and XPD-GFP do not interact with each other in the cytoplasm of living cells, despite the fact that these proteins do interact in the context of Transcription/repair factor IIH (TFIIH) [29]
TFIIH is implicated in a multitude of DNA-transactions, ranging from basal and activated RNA polymerase II transcription, to ribosomal RNA gene expression, participation in global genome Nucleotide excision repair (NER) (GG-NER) and transcription-coupled NER (TC-NER), and possibly cell-cycle regulation

Summary

Introduction

A network of distinct DNA-repair systems collectively removes most injuries and safeguards the stability of the genome [1]. The DNA helix is opened by the bi-directional helicase function of TFIIH. Within TC-NER, the stalled RNA polymerase likely first detects lesions whereas the Cockayne syndrome A and B proteins play roles in the early steps of this process. The GG-NER-specific complexes (xeroderma pigmentosum complementation group C [XPC] in complex with the human homologue of Rad23 [hHR23B/A], and xeroderma pigmentosum complementation group E [XPE/ DDB2] in complex with the UV-damaged DNA binding protein 1, [UV-DDB1]) recognize lesions at any position in the genome [5].

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Conclusion