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. 2006 Jun;4(6):e156.
doi: 10.1371/journal.pbio.0040156. Epub 2006 May 9.

Dynamic interaction of TTDA with TFIIH is stabilized by nucleotide excision repair in living cells

Giuseppina Giglia-Mari  1 , Catherine Miquel, Arjan F Theil, Pierre-Olivier Mari, Deborah Hoogstraten, Jessica M Y Ng, Christoffel Dinant, Jan H J Hoeijmakers, Wim Vermeulen
Affiliations

Dynamic interaction of TTDA with TFIIH is stabilized by nucleotide excision repair in living cells

Giuseppina Giglia-Mari et al. PLoS Biol. 2006 Jun.

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.

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Figures

Figure 1
Figure 1. Constructs and Functionality Assays
(A) Scheme of the TTD-A-GFP and XPD-GFP fusion proteins. (B) UV survival using wt VH10 cells (red diamonds), TTD1BRSV (TTD-A) cells (light blue squares), XP6BESV (XP-D) cells (pink squares), TTD1BRSV-expressing TTDA-GFP cells (green squares), and XP6BESV-expressing XPD-GFP cells (blue triangles). The percentage of surviving cells is plotted against the applied UV-C dose (J/m2). (C) Immunoprecipitation: Left panel shows that the XPB TFIIH subunit (by monoclonal anti-XPB detection) co-precipitates with anti-GFP from extracts of TTDA-GFP-expressing fibroblasts (lane 1) and not from TTD1BRSV (TTD-A) fibroblast whole-cell extracts (lane 2). Right panel shows that TTDA-GFP (detected with anti-GFP monoclonal) co-precipitates with the core TFIIH component p44 from extracts from TTDA-GFP-expressing fibroblasts (lane 1) and not in whole-cell extracts from TTD1BRSV (TTD-A) fibroblasts (lane 2). (D) Immunoprecipitation using polyclonal anti-GFP. Also the XPB TFIIH subunit co-precipitated with XPD-GFP (using anti-GFP) in extracts from XPD-GFP-expressing fibroblasts (lane 1), but not from XP6BESV (XP-D) fibroblasts (lane 2). (E) Immunofluorescence of a mixed population of TTD-A cells (label 2) and TTDA-GFP-expressing TTD-A cells. Cells expressing TTDA-GFP (right panel) showed an increased level of XPB (left panel), compared to TTD-A cells. (F) Immunofluorescence of a mixed population of VH10 (wt) cells and TTDA-GFP-expressing TTD-A cells. Cells expressing TTDA-GFP (right panel) exhibit a similar expression level of XPB (left panel) as wt cells.
Figure 2
Figure 2. Localization of TTDA-GFP and XPD-GFP
(A) Confocal image of a TTDA-GFP-expressing cell. (B) Confocal image of a XPD-GFP-expressing cell. (C) Confocal image of a XPB-GFP-expressing cell. (D) Immunoblot probed with anti-GFP monoclonal antibody of TTD1BRSV (TTD-A) fibroblasts stably expressing TTDA-GFP (lane 1), XP6BESV (XP-D) transformed fibroblasts stably expressing XPD-GFP (lane 3), and MRC5SV (wt) transformed fibroblasts expressing free GFP (lane 2 and 4).
Figure 3
Figure 3. Mobility of TTDA-GFP and XPD-GFP in the Cytoplasm and in the Nucleus Determined by FRAP
(A) FRAP analysis of XPD-GFP (blue line), TTDA-GFP (red line), and free GFP (green line) residing in the cytoplasm. Inset shows increased time resolution of the curves with error bars. (B) FRAP analysis of XPB-GFP (pink line), XPD-GFP (blue line), TTDA-GFP (red line), and free GFP (green line) in the nucleus. Relative fluorescence (fluorescence post-bleach divided by fluorescence pre-bleach) plotted against time in seconds. Inset shows increased time resolution of the curves with error bars. The p-value has been calculated for GFP and TTDA-GFP datasets.
Figure 4
Figure 4. FRAP_abc
(A) TTDA-GFP-expressing fibroblast without treatment (left panel) and after (right panel) applying several high laser pulses in the cytoplasmic compartment (see Materials and Methods for details). (B) TTDA-GFP mobility in the cytoplasm (green line), in the nucleus without bleaching the cytoplasm (red line), in the nucleus after bleaching the cytoplasmic fraction (blue line), and XPB-GFP mobility in the nucleus (pink line). The p-value has been calculated for cytoplasmic and nuclear mobility curves of TTDA-GFP. (C) XPD-GFP mobility in the cytoplasm (green line), in the nucleus without bleaching the cytoplasm (red line), in the nucleus after bleaching the cytoplasmic fraction (blue line), and XPB-GFP mobility in the nucleus (pink line). The p-value has been calculated for cytoplasmic and nuclear mobility curves of XPD-GFP. (D) FRAP_abc on XPB-GFP (pink line), XPD-GFP (blue line), TTDA-GFP (red line), and free GFP (green line). Inset shows increased time resolution of the curves with error bars.
Figure 5
Figure 5. Mobility of TTDA-GFP and XPD-GFP after UV Irradiation
(A) FRAP_abc of TTDA-GFP expressing cells untreated (blue line) and treated with 8J/m2 UV-C (red line). Error bars are included in the curves, and the p-value has been calculated for the two distinct datasets. (B) FRAP_abc of XPD-GFP-expressing cells untreated (blue line) and treated with 8J/m2 UV-C (red line). For each line at least 20 different cells were measured. Error bars are included in the curves, and the p-value has been calculated for the two distinct datasets. (C) Example of FRAP on local damage. A TTDA-GFP-expressing cell (left panel) containing a locally inflicted UV-damaged spot (shown by the white arrow). The locally damaged area is bleached by applying a high-pulse laser beam (middle panel), and the subsequent recovery of fluorescence is followed in time (right panel). (D) Curves of FRAP on local damage of TTDA-GFP- (red line), XPD-GFP- (blue line), and XPB-GFP-(green line) expressing cells. FLD, fluorescence measured on local damage; FNLD, fluorescence measured on untreated areas. Error bars are included in the curves.
Figure 6
Figure 6. Mobility of XPB-GFP, TTDA-GFP after Transcription Inhibition
(A) FRAP_abc of XPB-GFP-expressing cells untreated (blue line) and treated with dRB (see Materials and Methods) (red line). Inset shows increased time resolution of the curves with error bars. The p-value has been calculated for the two distinct datasets. (B) FRAP_abc of TTDA-GFP-expressing cells untreated (blue line) and treated with dRB (red line). Inset shows increased time resolution of the curves with error bars. The p-value has been calculated for the two distinct datasets.
Figure 7
Figure 7. Comparison of Accumulation of NER Proteins on Local UV Damage and on Localized ActD-Photosensitized Laser Damage
(A–D) Left panel, local DNA damage infliction by UV-C irradiation through a micro-porous filter. Right panel, local laser-induced (488 nm) DNA lesions by photo-sensitization of ActD. (A) XPC-GFP-expressing cells showing accumulation at local UV-damaged area (left panel) and laser induced ActD-damaged area (right panel). (B) XPB-GFP expressing cells showing accumulation at local UV-damaged area (left panel) and laser induced ActD-damaged area (right panel). (C) TTDA-GFP expressing cells showing only accumulation on local UV-damaged area (left panel) but not on laser induced ActD-damaged area (right panel); drawn rectangle corresponds with the irradiated area. (D) GFP-XPA expressing cells showing only accumulation on local UV-damaged area (left panel) but not on laser induced ActD-damaged area (right panel); drawn rectangle corresponds with the irradiated area.
Figure 8
Figure 8. Model of TTDA Binding to TFIIH during Transcription, NER, and Abortive NER
Schematic representation of a mammalian cell with the nucleus in gray and nuclear pores simplified by holes in the membrane. TTDA is represented as a green sphere, TFIIH is depicted as an orange ellipse, and XPC is illustrated as a yellow ellipse. Arrows indicate equilibrium (passage through) over the nuclear pores and equilibrium between different TTDA and/or TFIIH molecules. Colored arrows show the changes in the equilibrium after DNA-damage induction or ActD treatment. In “NER-induction” (right, upper), the UV lesion is depicted as a lightning-sign, in “Abortive NER” (right, bottom), ActD is depicted as a blue trapezoid, and the red cross represents the inhibition of transcription.
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