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In vivo interactions of TTDA mutant proteins withinTFIIH

Julie Nonnekens1,2, Stephanie Cabantous2,3,4, Joris Slingerland1,2, Pierre-Olivier Mari1,2 andGiuseppina Giglia-Mari1,2,*1CNRS, IPBS, 205 route de Narbonne, 31077 Toulouse, France2Universite de Toulouse, UPS, 118 route de Narbonne, 31062 Toulouse, France3INSERM UMR1037-Cancer Research Center of Toulouse, Toulouse, France4Institut Claudius Regaud, 20–24 rue du pont Saint-Pierre, 31052 Toulouse, France

*Author for correspondence (mari@ipbs.fr)

Accepted 9 May 2013Journal of Cell Science 126, 3278–3283� 2013. Published by The Company of Biologists Ltddoi: 10.1242/jcs.126839

SummaryTrichothiodystrophy group A (TTD-A) patients carry a mutation in the transcription factor II H (TFIIH) subunit TTDA. Using a novel in

vivo tripartite split-GFP system, we show that TTDA interacts with the TFIIH subunit p52 and the p52-TTDA-GFP product is

incorporated into TFIIH. p52-TTDA-GFP is able to bind DNA and is recruited to UV-damaged DNA. Furthermore, we show that twopatient-mutated TTDA proteins can interact with p52, are able to bind to the DNA and can localize to damaged DNA. Our findings givenew insights into the behavior of TTDA within the context of a living cell and thereby shed light on the complex phenotype of TTD-Apatients.

Key words: Protein–protein interactions, TFIIH, Trichothiodystrophy, Tripartite split-GFP, TTDA

IntroductionNucleotide excision repair (NER) safeguards the stability of the

genome by removing a broad range of helix-distorting lesions,

including UV-induced lesions (Hoeijmakers, 2009). One of the

NER key players is transcription factor II H (TFIIH). After

damage recognition, the DNA helix is opened by the TFIIH bi-

directional helicase function, to allow incision of the damaged

strand, gap filling and ligation (Palomera-Sanchez and Zurita,

2011). Besides NER, TFIIH is also essential for RNA polymerase

I and II transcription and cell cycle regulation (Egly and Coin,

2011). TFIIH is composed of 10 proteins, consisting of a core-

complex (XPB, XPD, p62, p52, p44, p34 and TTDA) and a

cyclin activating kinase-subcomplex (CDK7, MAT1 and cyclin

H) (Giglia-Mari et al., 2004).

Hereditary mutations in TFIIH are associated with severe features,

as presented by several photo-sensitive syndromes, such as

trichothiodystrophy (TTD) (Lehmann, 2003). TTD is a rare

autosomal disorder and patients suffer from premature aging,

progressive mental and growth retardation, ichthyosis and brittle

hair and nails (Stefanini et al., 2010). Photosensitive TTD is an

exclusive TFIIH-related syndrome and three genes have been found

mutated in TTD patients: XPB (Weeda et al., 1997), XPD (Broughton

et al., 1994; Stefanini et al., 1993) and TTDA (Giglia-Mari et al.,

2004). Mutations in any of these three genes cause a decrease by up to

70% in TFIIH cellular concentration (Botta et al., 2002).

TTDA is an 8 kDa protein that stabilizes the steady-state level

of TFIIH (Giglia-Mari et al., 2004; Ranish et al., 2004; Vermeulen

et al., 2000). TTDA is essential for NER by stabilizing TFIIH on

the lesions (Giglia-Mari et al., 2006) and by allowing the

subsequent NER factors to localize to the damage (Coin et al.,

2006; Theil et al., 2011). TTD group A (TTD-A) patients carry a

mutation in TTDA and display a relatively mild TTD phenotype

(Giglia-Mari et al., 2004). Recently, it was shown that in TTD-A

cells, repair is not completely abolished, but progresses slowly

(Theil et al., 2011). This also explains the mild UV sensitivity of

the patient cells (Giglia-Mari et al., 2004; Vermeulen et al., 2000).

Three mutations within the TTDA gene of non-related TTD-A

patients have been detected (Giglia-Mari et al., 2004). Siblings

TTD13PV and TTD14PV carry a homozygous mutation in the

start codon, converting the methionine into a threonine (M1T).

This will lead to either a complete loss of protein synthesis or to the

production of an N-terminal truncated polypeptide when a

downstream start codon at amino acid position 16 is used.

Patient TTD99RO carries a homozygous transition at amino acid

56, converting an arginine into a stop codon (R56X); this truncates

the protein at the C-terminal end by 23%. Patient TTD1BR has one

allele identical to that of patient TTD99RO and the other allele has

a transition at amino acid 21 that converts a leucine into a proline

(L21P). Intriguingly, despite the rather diverse mutations, a similar

appearance of the clinical features is observed among TTD-A

patients (Giglia-Mari et al., 2004).

In vitro studies have given knowledge about the binding

behavior of TTDA to the TFIIH subunit p52. For now, no

mutations in p52 have been reported that are linked to human

disease. However, defective p52 in Drosophila melanogaster can

generate TTD and cancer-like phenotypes (Fregoso et al., 2007).

Structural studies on yeast TTDA and p52 have shown that the C-

terminal of p52 anchors TTDA and forms a heterodimer (Kainov

et al., 2010; Kainov et al., 2008). Further in vitro studies showed

that via this interaction with p52, TTDA is able to regulate XPB

ATPase activity (Coin et al., 2006). The interaction between

TTDA and p52 was validated by in vitro pull-down experiments,

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but has never been demonstrated in vivo. Here, we use a novel

tripartite split-GFP system to study the TTDA and p52 interaction

in its most relevant environment, the living cell. This three-body

split-GFP system uses two b-strands of GFP, b-strand 10 (GFP10)

and b-strand 11 (GFP11) as fusion tags of the proteins of interest.

A large complementary detector fragment corresponds to b-strand

1–9 (GFP1-9). When the tagged proteins interact, GFP10 and

GFP11 are tethered and are then capable of self-associating with

the complementary GFP1-9 fragment to reconstitute full-length

GFP; the cell will exhibit a green fluorescent signal (Fig. 1A). This

system has no background fluorescence and will not spontaneously

self-associate as previously found with the two body split-GFP 1-

10 and GFP11 (S. Cabantous, J.D. Pedelacq, H.B. Nguyen,

A. Chaudhary, K. Ganguly, F. Koraıchi, M.A. Lockard, G. Favre,

T.C. Terwilliger and G.S. Waldo, unpublished observations).

Using this novel system, we show that p52 interacts with TTDA

in vivo. This p52-TTDA-GFP product is incorporated in TFIIH,

can bind to the DNA and is recruited to an UV damaged region.

Our results also demonstrate that two of the TTDA patient-mutated

forms (TTDAL21P and TTDAR56X) can interact with p52 and that

they behave the same as wild-type TTDA. Additionally, we show

that the N-terminal truncated patient mutant (TTDAM1T) is not

able to interact with p52 but still can be part of TFIIH, although the

binding capacity is diminished. Our findings give new insights into

TTDA behavior in binding to and functioning with TFIIH within

the context of a living cell; not only in the wild-type situation, but

also in relation to TTD-A patient mutations.

Results and DiscussionTTDA interacts with p52 in vivo

To investigate how TTDA interacts with TFIIH in the mostrelevant biological context, the living cell, we studied the binding

of TTDA and p52, previously shown in structural works (Kainovet al., 2008), using the tripartite split-GFP system (Fig. 1A). TheGFP1-9 fragment was stably expressed in human transformed

fibroblasts and we have produced expressing systems with p52 C-terminally tagged with GFP11 (p52-GFP11) and TTDA (wild-

type and mutated) N-terminally tagged with GFP10 (GFP10-TTDA) (supplementary material Fig. S1). Previously, it was

shown that tagged TTDA is biologically active (Giglia-Mari et al.,2006). Using the tripartite split-GFP technology, we demonstrate

that TTDA interacts with p52 in vivo (Fig. 1B). Along themanuscript, this protein complex will be called p52-TTDA-GFP.

TFIIH is a nuclear protein complex, however, the order of

protein assembly and the cellular compartments in which thisassembly takes place are not fully known. Interestingly, the GFP

fluorescence produced by p52-TTDA-GFP was not only observedin the nucleus, but also in the cytoplasm, indicating that the

interaction between p52 and TTDA already starts in this cellularcompartment (Fig. 1B).

To verify if the observed GFP reconstitution was not due to afalse-negative event, we first tested if deleting the TTDA-interacting domain from p52 (Kainov et al., 2008) [p52(DC-

term)-GFP11] hindered its interaction with TTDA. Indeed,p52(DC-term) is not able to interact with TTDA as shown by the

Fig. 1. The tripartite split-GFP system and p52 and

TTDA interactions. (A) The three-body split-GFP

system. When protein A is fused to GFP10 (yellow) and

B is fused to GFP11 (orange), interaction between A:B

drives GFP10 and GFP11 in close proximity. They are

then capable of associating with the GFP1-9 fragment and

reconstituting the GFP fluorophore (S. Cabantous, J.D.

Pedelacq, H.B. Nguyen, A. Chaudhary, K. Ganguly, F.

Koraıchi, M.A. Lockard, G. Favre, T.C. Terwilliger and

G.S. Waldo, unpublished observations). (B) MRC5-SV +

GFP1-9 cells transiently co-express GFP10-TTDA (wild-

type or mutated) and p52-GFP11. Reconstitution of GFP

was visualized for TTDAWT (first panel), TTDAL21P

(second panel) and TTDAR56X (third panel) and no GFP

reconstitution was visualized for TTDAM1T (fourth

panel). Scale bars: 5 mm. (C) Fluorescent recovery after

photobleaching (FRAP) on XPB-GFP and p52-TTDAWT-

GFP fusion products. (D) FRAP on p52-TTDA-GFP

fusion products: TTDAWT, TTDAR56X and TTDAL21P.

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inability to reconstitute the GFP fluorophore (supplementarymaterial Fig. S2A,B). Meanwhile, co-expression of p52(DC-term)-

GFP11 with GFP1-10 (a large complementary detector fragmentcorresponding to b-strand 1–10) did show GFP fluorescence(supplementary material Fig. S2C,D), indicating that p52(DC-

term)-GFP11 is properly expressed. Furthermore, we measured thepossible interaction between both TTDA and p52 with XPG. XPG isan endonuclease that closely functions together with TFIIH during

NER and binds to TFIIH via the XPD subunit (Ito et al., 2007). Nointeraction of XPG with TTDA or p52 has been described in theliterature. Indeed, when we expressed both plasmids (XPG-GFP10with p52-GFP11 or XPG-GFP10 with GFP11-TTDA) in the GFP1-

9 cell line, we did not observe reconstitution of the GFP fluorophore(supplementary material Fig. S2E,F).

Mutated TTDA can interact with p52

To investigate whether mutant TTDA could interact with p52, weco-expressed p52-GFP11 with the GFP10-TTDA patient mutant

constructs (supplementary material Fig. S1) and verified thereconstitution of the GFP fluorophore. In vitro studies showedthat the solubility of the TTDA mutants L21P and R56X was

drastically affected when purified. Analysis of the crystallographic

structure suggested that the L21P mutation may disrupt theconformation of TTDA which could result in protein unfolding(Hashimoto and Egly, 2009). We observed GFP fluorescence in the

nucleus and the cytoplasm for both TTDA mutants, just as for wild-type TTDA (Fig. 1B). This indicates that, despite their mutations,these two forms of TTDA are able to interact with p52 in vivo.

Previously, it was shown in vitro that an N-terminally truncatedTTDA protein without the first 14 amino acids (TTDADN14) isunable to interact with p52 (Zhou et al., 2007). Indeed, we do not

observe interaction between TTDAM1T (lacking the first 15 aminoacids of TTDA) and p52 (Fig. 1B). Our results confirm that the N-terminal region of TTDA is essential for interaction with p52 in

vivo. Moreover, we show that the C-terminal region of TTDA

[which was also suggested in structural studies to interact with p52(Kainov et al., 2008)], is not sufficient for TTDA binding to p52 in

vivo, without the presence of the N-terminal domain.

TTDA does not dimerize in vivoIt was proposed from structural studies that TTDA has the ability to

self-associate at the N-terminal region (Vitorino et al., 2007). Toinvestigate this dimerization in vivo, we co-expressed GFP10-TTDA and GFP11-TTDA in the GFP1-9 cells. Interestingly, we did

not observe reconstitution of the GFP fluorophore, indicating thatTTDA cannot form a dimer in vivo (supplementary material Fig. S3).

p52-TTDA-GFP is integrated in TFIIHUnder normal conditions, p52 is a stable subunit of core-TFIIH andTTDA shuttles between a free fraction (nuclear and cytoplasmic)

and a fraction bound to TFIIH (Giglia-Mari et al., 2006). In thetripartite split-GFP system, the proteins of interest are irreversiblycoupled to each other via GFP. To investigate whether thisirreversible coupling between p52 and TTDA hinders p52-TTDA-

GFP to be integrated into core-TFIIH, we measured the mobility ofp52-TTDA-GFP and compared it to the core-TFIIH mobility byfluorescent recovery after photobleaching (FRAP) (Giglia-Mari

et al., 2006; Hoogstraten et al., 2002). Briefly, a narrow stripspanning the nucleus was photobleached and recovery offluorescence was measured in order to determine the mobility of

the proteins. XPB is a TFIIH subunit that only resides within core-TFIIH and fluorescent tagged XPB (XPB-GFP) can therefore beused to measure its mobility (Hoogstraten et al., 2002). Our results

show that the mobility of p52-TTDA-GFP is comparable to the oneof XPB-GFP measured under the same conditions (Fig. 1C). Thesmall difference might be accounted for by the fact that within theexpressing cells also endogenous p52 and TTDA proteins are

present and the p52-TTDA-GFP complex therefore has to competefor its placement within TFIIH. However, the FRAP data suggestthat p52-TTDA-GFP is able to integrate into core-TFIIH (p52-

TTDA-GFP-TFIIH).

We further investigated whether the p52-TTDAL21P-GFP andp52-TTDAR56X-GFP complexes were able to integrate TFIIH by

comparing their mobility with the one measured for the p52-TTDAWT-GFP. Fig. 1D shows that all complexes have the samemobility, indicating that also these mutant complexes are

integrated into TFIIH.

p52-TTDA-GFP binds to DNA

TTDA has been found to be a repair-specific TFIIH subunit in

vitro and TTDA shuttling in and out of TFIIH might influenceTFIIH activity in NER (Coin et al., 2006). Since in the tripartite

Fig. 2. p52-TTDA-GFP in transcription and DNA repair. (A–C) FRAP

studies on (A) p52-TTDAWT-GFP, (B) p52-TTDAL21P-GFP and (C) p52-

TTDAR56X-GFP fusion products. Cells were measured untreated (NT), 30–

90 min after exposure to 16 J/m2 UV-C (+UV) and after 6–7 hours treatment

with DRB (+DRB). Error bars are s.e.m. (D) Accumulation of p52-TTDA-

GFP complexes on locally damaged DNA (arrows): TTDAWT (left panel)

TTDAL21P (middle panel) and TTDAR56X (right panel). Scale bars: 5 mm.

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split-GFP system TTDA and p52 are irreversibly coupled, we

verified whether the p52-TTDA-GFP-TFIIH complex is able to

bind to DNA during transcription and repair. We measured the

mobility of p52-TTDA-GFP-TFIIH after treatment with the

transcription inhibitor DRB (5,6-dichloro-1b-D-ribofuranosyl

benzimidazole) or global UV-C irradiation. Transcription

inhibition increases the mobility of TFIIH by releasing the

complex from the chromatin while UV treatment triggers NER

in which TFIIH is bound to the DNA more tightly (Giglia-Mari

et al., 2006). We confirmed this in our experimental setting by

measuring the mobility of XPB-GFP after UV or DRB treatment.

Indeed, we observed a decreased mobile XPB fraction after UV

irradiation and a higher mobility after DRB treatment.

Furthermore, cells expressing XRCC4-GFP (non-homologous

end joining repair factor), do not show a response to UV and

DRB treatment (supplementary material Fig. S4), indicating the

specificity of the FRAP experiments.

Our results show that p52-TTDA-GFP-TFIIH has an increased

mobility after transcription inhibition, indicating that under normal

conditions, this version of TFIIH can bind to DNA on promoters.

Furthermore, its mobility is reduced after UV exposure (Fig. 2A),

meaning that the complex is most likely recruited to the damaged

DNA in order to function in DNA repair.

Next, we tested whether p52-TTDA-GFP-TFIIH was able to

localize on damaged DNA. We have locally irradiated a small

region of the nucleus using a multiphoton laser (Mari et al., 2006)

and a clear fluorescent spot appeared at the laser targeted area,

indicating that p52-TTDA-GFP-TFIIH is able to accumulate on

damaged DNA (Fig. 2D). Our results suggest that TFIIH can

bind to the DNA during transcription and repair even when

TTDA is not able to shuttle in and out of the complex.

Furthermore, binding of p52-TTDA-GFP-TFIIH to the DNA

and recruitment to damaged DNA is not only observed for p52-

TTDAWT-GFP, but also for the mutants p52-TTDAL21P-GFP and

p52-TTDAR56X-GFP (Fig. 2B–D).

TTDAM1T interacts with TFIIH independently of p52

We showed that TTDAM1T is not able to interact with p52

(Fig. 1B) making it impossible to study the behavior of this

mutant with the tripartite split-GFP system. In order to examine

Fig. 3. TTDAM1T-GFP interaction with TFIIH.

(A) TTDAM1T-GFP localization. Scale bar: 5 mm.

(B) TTDAM1T-GFP-expressing cell showing the location of

the locally damaged area (circle). Scale bar: 5 mm.

(C) FRAP_abc curves showing the mobility of TTDAWT-GFP

and TTDAM1T-GFP. Error bars are depicted as s.e.m.

(D) Quantification of the cytoplasm fluorescence after

bleaching the cytoplasm, before and after FRAP_abc. Post-

FRAP fluorescent signal is normalized to pre-FRAP signal.

Error bars are depicted as s.e.m. **P,0.01.

Fig. 4. Model for TTDA interactions with TFIIH. (A) TTDAWT,

(B) TTDAL21P and (C) TTDAR56X have the ability to interact with p52 and

can thereby stimulate the XPB ATPase activity. However, the binding of

(B) TTDAL21P and (C) TTDAR56X to p52 does not stabilize the TFIIH steady-

state level. (D) TTDAM1T cannot interact with p52 and XPB ATPase activity

is not stimulated.

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if, despite the mutation, TTDAM1T is still incorporated into

TFIIH and is recruited to the damaged DNA, we produced

TTDAM1T-GFP-expressing cells. TTDAM1T-GFP fluorescence is

observed in both the nucleus and the cytoplasm (Fig. 3A) just as

TTDAWT-GFP, which is part of functional core-TFIIH (Giglia-

Mari et al., 2006). We induced local DNA damage with a

multiphoton laser (Mari et al., 2006), but no accumulation of

TTDAM1T-GFP at the damaged region was observed (Fig. 3B).

Absence of accumulation on damaged DNA suggests that

TTDAM1T is not able to bind to TFIIH or that detection is

below threshold. To discriminate between these two possibilities,

we compared the mobility of TTDAWT-GFP and TTDAM1T-GFP

by FRAP after bleaching the cytoplasm (FRAP_abc) (Giglia-

Mari et al., 2006). Our results show that TTDAM1T is slightly

more mobile than TTDAWT (Fig. 3C) but certainly less mobile

than an unbound TTDA protein (Giglia-Mari et al., 2006). Due to

fast exchange of TTDA between the nucleus and cytoplasm the

FRAP curves decline quickly over time and the decline is quicker

in the TTDAM1T compared to TTDAWT (Fig. 3C, zoom 12–

16 seconds). This faster exchange is also observed by measuring

the fluorescence in the cytoplasm after bleaching the cytoplasm

just before and directly after the FRAP experiment (on average

30 seconds difference). Both proteins (WT and mutant) quickly

shuttle into the cytoplasm, however this shuttling (measured by

an increase of fluorescence in the cytoplasm) is significantly

higher for TTDAM1T (Fig. 3D).

Altogether, our results indicate that without its N-terminal

region, TTDA cannot bind to p52 (tripartite split-GFP

experiments), but this mutant is still able to interact with

TFIIH, however not as tightly as TTDAWT (FRAP_abc data).

Indeed, in vitro studies suggested that TTDA could interact with

the core-TFIIH subunit XPD (Coin et al., 2006). This interaction

is apparently not stable or strong enough to see TTDAM1T-GFP

accumulation on locally damaged DNA, but is sufficient to

reduce the mobility of this mutant compared to unbound TTDA.

Concluding remarks

With the use of the tripartite-split GFP system, we show that p52 and

TTDA interact within the living cell. Interestingly, this interaction is

observed not just between p52 and wild-type TTDA, but also

between p52 and two forms of patient-mutated TTDA: TTDAL21P

and TTDAR56X. The p52-TTDA-GFP complex (WT and mutant)

can be integrated into TFIIH, is recruited to the chromatin during

transcription and accumulates on damaged DNA.

In contrast, the N-terminal truncated form (M1T) totally lacks

the ability to bind p52. Despite the difference in p52 binding

capacity, all patients with TTD-A have the same reduced TFIIH

steady-state level of ,30% (Giglia-Mari et al., 2004; Vermeulen

et al., 2000). This means that, even though TTDAL21P and

TTDAR56X retain the ability to bind p52, this is not enough to

further stabilize TFIIH levels. However, despite a similar TFIIH

steady state level, patient cells with TTDAM1T are slightly more

sensitive to UV exposure and have less overall NER activity

(Giglia-Mari et al., 2004). We suggest that the binding of

TTDAL21P and TTDAR56X to p52 still enables p52 to stimulate

the ATPase-activity of XPB which is necessary for proper NER

(Coin et al., 2007). We propose a model for TTDA interactions

with TFIIH in Fig. 4. Because the three patient mutant TTDA

proteins, still interact in vivo with TFIIH (via p52 or

independently of p52), we can hypothesize that these proteins

keep some functionality and that they cannot be considered asnull mutants.

Altogether, our observations give a further insight into thebehavior of TFIIH in TTD-A patients and thereby help with

the understanding of the molecular mechanisms behind thepathophysiology of this severe disease.

Materials and MethodsCell culture and treatment

SV40-immortalized human fibroblasts were cultured in 1:1 mixture of DMEM andHam’s F10 (Lonza) with antibiotics and 10% fetal bovine serum, at 37 C and 5%CO2. HT1080 + XRCC4-GFP cells were cultured in DMEM with antibiotics and10% fetal bovine serum, at 37 C and 5% CO2.

Treatment: 6–7 hours 100 mg/mL DRB (5,6-dichloro-1b-D-ribofuranosylbenzimidazole) or 16 J/m2 UV-C irradiation and imaging after 30–90 min.

Plasmids

GFP1-9 was cloned into pEGFP-N3 between NheI and NotI sites. p52 and werecloned into pcDNA derived plasmids containing the GFP10 or GFP11 sequences.p52 was cloned into NotI and ClaI sites of pcDNA_GFP11 and TTDA into BspEIand XbaI sites of pcDNA_GFP10 (S. Cabantous, J.D. Pedelacq, H.B. Nguyen, A.Chaudhary, K. Ganguly, F. Koraıchi, M.A. Lockard, G. Favre, T.C. Terwilligerand G.S. Waldo, unpublished observations). TTDAM1T-GFP has been created byremoving the first 15 amino acids of TTDA from TTDAWT-GFP in pEGFP-N1(Giglia-Mari et al., 2006) using a site directed mutagenesis approach.

Transfection and electroporation

The MRC5-SV cells were transfected with the GFP1-9 and TTDA-GFP (WT orM1T) plasmids using JetPEI transfection reagent (Polyplus transfection) andselected with geneticin (Gibco) to obtain stable cell lines. MRC5-SV + GFP1-9cells were co-electroporated with the GFP10 and GFP11 plasmids and left toincubate for 24–48 hours before imaging. Electroporation was performed with4 mm cuvettes in a Gene Pulser Xcell electroporator (Bio-Rad) with a voltage of250 V and capacity of 950 mF.

Confocal imaging

Imaging experiments were performed on a LSM 710 NLO confocal laser scanningmicroscope (Zeiss), using a 406/1.3 objective and under a controlled environment(37 C, 5% CO2). Images were analyzed with ImageJ software.

FRAP

A narrow strip spanning the nucleus of a fluorescent living cell was monitored every20 or 200 msec at 1% laser intensity (30 mW argon laser, current set at 6.5 A,488 nm line) and photobleached at maximum laser intensity. Fluorescence recoverywas monitored up to 16 seconds. For FRAP_abc, mobility measurements wereobtained after bleach pulses (100% laser intensity) were applied to the cytoplasm for,1 minute (Giglia-Mari et al., 2006; Hoogstraten et al., 2002).

For the analysis ZEN software (Zeiss) was used. All FRAP data were normalizedto average pre-bleached fluorescence after background signal removal. FRAP curvesare an average of ten measured cells and error bars represent the s.e.m.

Local damage

DNA damage was induced with a tunable near-infrared pulsed laser (CameleonVision II, Coherent Inc., USA). A small circular area (4 mm) was targeted for34 milliseconds (single scan iteration at 800 nm, 60% power output). The cell wasimaged ,60 seconds after damage induction (Mari et al., 2006).

Statistics

We determined P-values by a 2-tailed Student’s t-test, assuming equal variance:**P,0.01. We considered differences between groups significant at P,0.05.

AcknowledgementsWe would like to thank P. Calsou and S. Britton (IPBS, CNRS,Toulouse, France) for the XRCC4-GFP cells. We are grateful to allthe members of the team of GGM for helpful discussions. Theauthors declare that they have no conflict of interest.

Author contributionsG.G.-M., S.C. and J.N. designed the experiments. J.N. performed theexperiments, G.G.-M. and J.N. analyzed the data. J.S. assisted withcloning and P.-O.M. assisted with microscopy. G.G.-M. and J.N.wrote the paper.

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FundingThis work has been supported by Region Midi-Pyrenees [grant no.08017067 to J.N.], Fondation pour la Recherche Medicale [grant no.FDT20120925285/086257 to J.N.], Agence National de la Recherche[grant no. ANR10-BLAN-123101-01 to J.N., J.S., G.G.-M.] andAction Thematique et Incitative sur Programme plus [ATIPAVENIR 2011 Cancer 201204/080192 to J.N., J.S., P.-O.M.,G.G.M.]. This work also benefited from the TRI Optical ImagingPlatform at IPBS (Genotoul, Toulouse, France) supported by grantsfrom the Region Midi-Pyrenees (CPER), the Grand Toulousecommunity, the ARC [ARC Equipment No. 8505], the Centrenational de la recherche scientifique (CNRS) and the EU through theFonds Europeen de Developpement Regional program.

Supplementary material available online at

http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.126839/-/DC1

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Interactions of TTDA within TFIIH 3283