DNA-damaging agents constantly challenge the integrity of DNA. A network of distinct DNA-repair systems collectively removes most injuries and safeguards the stability of the genome [
]. Nucleotide excision repair (NER) is a DNA repair mechanism capable of removing different structurally unrelated DNA helix-distorting lesions, including ultraviolet light (UV)-induced lesions and bulky chemical adducts. NER requires the concerted action of ˜25 proteins in a coordinated multi-step process [
]. After initial damage recognition, the DNA helix is opened by the bi-directional helicase function of TFIIH. Subsequently, four proteins (xeroderma pigmentosum complementation group G protein [XPG], xeroderma pigmentosum complementation group A protein [XPA], replication protein A, and excision repair cross complementing group 1 protein [ERCC1] in complex with xeroderma pigmentosum group F [XPF]) are recruited to stabilize the open intermediate, verify the damage, and incise the damaged strand 3′ and 5′ at some distance from the injury [
]. Finally, the resulting gap is filled by repair replication and sealed by ligation. Within NER, two lesion recognition pathways are operational: transcription-coupled NER (TC-NER) and global genome NER (GG-NER) [
]. TC-NER is dedicated to lesions that block RNA polymerase II elongation. Within TC-NER, the stalled RNA polymerase likely first detects lesions whereas also 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 [
]. GG-NER mainly protects against damage-induced mutagenesis and can thus be considered as a cancer-preventing process, whereas TC-NER primarily promotes cellular survival, and therefore may prevent aging [
]. Hereditary NER deficiency is associated with severe clinical features as presented by three photo-sensitive disorders: the cancer-prone syndrome xeroderma pigmentosum (XP) and the neurodevelopmental conditions Cockayne syndrome and trichothiodystrophy (TTD)[
]. TTD is a premature aging syndrome, with the hallmark feature of brittle hair and nails, ichthyosis, and progressive mental and physical retardation also seen in Cockayne syndrome. Within photo-sensitive TTD, three TFIIH coding genes are implicated: xeroderma pigmentosum complementation group B (XPB) [
], xeroderma pigmentosum complementation group D (XPD) [
], and the newly identified protein termed “TTD group A” (TTDA) [
Besides GG-NER and TC-NER, TFIIH is also engaged in RNA polymerase II transcription initiation, RNA polymerase I transcription, activated transcription, and cell cycle regulation [
]. TFIIH consists of ten subunits, five of which (XPB, p62, p52, p44, and p34) form a tight core-complex, and the trimeric cyclin activating kinase-subcomplex (CDK7, MAT1, and cyclin H) is linked to the core via the XPD protein [
]. The recently identified 8-kDa TTDA protein [
] connects to the core via interactions with p52 and XPD . TFIIH harbors different enzymatic activities: two DNA-dependent ATPases, XPB and XPD, required for the helicase function [
], a protein kinase displayed by CDK7 [
], and the recently uncovered ubiquitin ligase activity of p44 [
]. Currently, the functions or possible enzymatic properties of the other TFIIH subunits including that of TTDA remain enigmatic.
Cells from patients with TTD-A have reduced steady-state levels of TFIIH, due to mutated
], suggesting that an important role of TTDA is to stabilize the entire TFIIH complex. The most striking feature of TTD-A cells is their reduced DNA repair activity. Intriguingly, the identified mutations, within the
gene of three non-related patients with TTD-A, lead either to the complete absence of the protein (mutation on the first ATG), or to non-functional truncated peptides, making TTDA the first TFIIH subunit for which a complete absence is compatible with life [
]. Despite these rather diverse mutations, a surprisingly similar expression of the clinical features is observed amongst the patients.
In order to investigate the participation of TTDA in DNA repair and transcription, we measured the differences in mobility of TTDA during repair and transcription in living cells by confocal imaging of a functional green fluorescent protein (GFP)-tagged TTDA (TTDA-GFP) expressed in TTDA-deficient transformed fibroblasts. To compare the mobility of TTDA with others TFIIH subunits, we also measured kinetic parameters of XPB-GFP and XPD-GFP in each of these processes.