TTD-A as a Repair-Specific TFIIH Subunit

Molecular Cell 21, 215–226, January 20, 2006 ª2006 Elsevier Inc.

DOI 10.1016/j.molcel.2005.10.024

p8/TTD-A as a Repair-Specific TFIIH Subunit

Fre´de´ric Coin,1,* Luca Proietti De Santis,1 Tiziana Nardo,2 Olga Zlobinskaya,1 Miria Stefanini,2 and Jean-Marc Egly1,* 1 Institut de Ge´ne´tique et de Biologie Mole´culaire et Cellulaire Centre National de la Recherche Scientifique Institut National de la Sante´ et de la Recherche Me´dicale Universite´ Louis Pasteur BP 163 67404 Illkirch Cedex C. U. Strasbourg France 2 Istituto di Genetica Molecolare Consiglio Nazionale delle Ricerche Via Abbiategrasso, 207 27100 Pavia Italy

Summary How subunits of the transcription/repair factor TFIIH cooperate to allow for the removal of DNA lesions or for the transcription of genes is crucial to understand the functioning of this complex. Here, we reveal that p8/TTD-A, the tenth subunit of TFIIH, has a critical role in DNA repair where it triggers DNA opening by stimulating XPB ATPase activity together with the damage recognition factor XPC-hHR23B. Fluorescent antibody labeling shows that such opening is needed for the recruitment of XPA to the site of the damage. By contrast, p8 is dispensable for RNA synthesis and doesn’t interfere with the transcriptional function of CAK, although both interact with the XPD subunit. Interestingly, p8 overexpression in TTD-XPD cells counteracts the detrimental effect of XPD mutations by restoring the cellular TFIIH concentration. These findings resolve the primary functions of p8 and unveil how TFIIH components specifically direct the complex toward repair or transcription. Introduction The understanding of genome expression requires the identification of the transcription and repair machinery components and the elucidation of the intricate connections between these two fundamental cellular processes. TFIIH consists of ten subunits and is resolved into a core and a CAK subcomplex bridged by the XPD polypeptide (Frit et al., 1999; Zurita and Merino, 2003). TFIIH is involved in nucleotide excision repair (NER) (Schaeffer et al., 1993) that removes a broad range of helix-distorting injuries: e.g., UV light-induced pyrimidine dimers and bulky chemical adducts. Two modes of DNA damage detection exist: the transcription-coupled repair (TC-NER) and the global genome repair (GG-NER)

*Correspondence: [email protected] (F.C.); egly@igbmc. u-strasbg.fr (J.-M.E.)

pathways. In TC-NER, an elongating RNA polymerase II (RNA Pol II) stalled by a lesion in the transcribed strand triggers efficient repair of the cytotoxic damages that block transcription (Hanawalt, 2002). Lesions anywhere in the genome are targets for GG-NER, initiated by the XPC/hHR23B complex (Sugasawa et al., 1998). Several pieces of evidence suggest that only some of the enzymatic activities and/or subunits of TFIIH are required for a given function of the complex. For instance, XPD helicase activity has a minor role in transcription but is necessary for removing DNA damages (Guzder et al., 1994a; Tirode et al., 1999). On the contrary, XPB activities catalyze both the opening of the DNA around the promoter to prime the nascent RNA and the opening of the DNA around a lesion to allow the excision of the damaged oligonucleotide (Evans et al., 1997; Guzder et al., 1994b; Holstege et al., 1996). Likewise, the cyclin-dependent kinase cdk7, a subunit of CAK, is required for the phosphorylation of the carboxy-terminal domain (CTD) of RNA Pol II, a crucial step for mRNA elongation (Schwartz et al., 2003). As part of TFIIH, cdk7 is also involved in hormone regulation through the phosphorylation of several transcriptional activators, including the nuclear receptors (Chen et al., 2000; Rochette-Egly et al., 1997). By contrast, the role of CAK in repair remains to be clarified (Araujo et al., 2000). Additionally, the yeast homolog of p44 possesses an E3 ubiquitin ligase activity important for the transcriptional response to DNA damage, but not for repair (Takagi et al., 2005). The multisubunit composition of TFIIH and the functional specialization of its components also permit the control of its engagement in transcription and repair through specific posttranslational modifications. Indeed, during mitosis, the phosphorylation of cdk7 by the mitotic cdc2/MPF kinase inhibits the transcriptional activity of TFIIH (Akoulitchev and Reinberg, 1998). Recently, we demonstrated that the phosphorylation of XPB controls TFIIH repair without interference in its transcription activity (Coin et al., 2004). Mutations in the XPB and XPD helicases of TFIIH give rise to xeroderma pigmentosum (XP) and trichothiodystrophy (TTD). These diseases exhibit a broad spectrum of clinical features, including photosensitivity of the skin due to defects in NER (Lehmann, 2003). Recently, we have identified p8/TTD-A (hereafter called ‘‘p8’’), the tenth subunit of TFIIH that is responsible for the third group of the photosensitive form of TTD (Giglia-Mari et al., 2004; Ranish et al., 2004). TTD patients present sulfur-deficient brittle hair and nails caused by a reduced level of cysteine-rich matrix proteins. Associated features include small stature, mental retardation, ichthyotic skin, unusual facial features, and photosensitivity (Itin et al., 2001). The concentration of TFIIH is reduced in TTD cells, including the TTD-A cells (Botta et al., 2002; Vermeulen et al., 2000), but this fact alone cannot explain the severe NER defects and the clinical symptoms of the patients that rather suggest an involvement of p8 in the transcription and repair activities of TFIIH. Here, we demonstrate the specific requirement of p8 in NER where it stimulates the opening of the DNA around

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the damage, a prerequisite for the arrival of XPA. We also investigate the crosstalk of p8 and CAK to regulate the activity of TFIIH in repair or transcription.

Results p8 Is Part of a TFIIH Functioning Both in Repair and Transcription To gain insight into the physical and functional engagement of p8 into the TFIIH complex, TFIIH from HeLa whole-cell extracts was immobilized on Ab-p44/protein A Sepharose (p44 is a subunit of TFIIH), treated with increasing KCl concentration, and eluted with the competitor peptide (Coin et al., 1999). A strong association of p8 to the complex is observed (Figure 1A). The Ab-p44 immunopurified TFIIH fraction (TFIIH/Ab-p44, Figure 1A, lane2) was next immunoprecipitated with a monoclonal antibody raised toward p8 (Ab-p8), and TFIIH/Ab-p8 was eluted with the competitor peptide. No TFIIH is found in the supernatant (Figure 1B, top, lanes 1 and 2), and TFIIH/Ab-p8 displays a similar amount of the distinct TFIIH subunits than TFIIH/Ab-p44 (lanes 1 and 3), indicating that all the cellular TFIIH contains the p8 subunit. Then, TFIIH/Ab-p44 and TFIIH/Ab-p8 were tested either in a dual incision assay (NER) containing the recombinant XPC-hHR23B, XPA, RPA, XPG, ERCC1/XPF factors, and a monomodified cis-platin plasmid (PtDNA) (Coin et al., 2004) or in a reconstituted transcription assay containing the recombinant TFIIB, TFIIF, TBP, TFIIE factors, the purified RNA Pol II, and the adenovirus major late promoter (AdMLP) template (Gerard et al., 1991). TFIIH/Ab-p8 and TFIIH/Ab-p44 exhibit similar DNA repair and transcription activities; no activity is found in the supernatant (Figure 1B, bottom). To determine the interacting partners of p8 within TFIIH, recombinant TFIIH subunits were tested for their interaction with bacterially produced and purified GST-p8 polypeptide. XPD and p52 are pulled down with GSTp8 (Figure 1C, lanes 8 and 10). These interactions were further confirmed by coexpression in baculovirus-infected insect cells (data not shown).

p8 Directs TFIIH Repair Activity To unravel the role of p8 in TFIIH functional activities, we purified, from baculovirus-infected insect cells, a recombinant TFIIH complex (IIH9) containing the nine subunits of TFIIH (XPB, XPD, p62, p52, p44, p34, cdk7, cyclH, and MAT1) (Tirode et al., 1999) without p8 (Figure 2A, top, lane 2). Strikingly, the repair activity of IIH9 is much lower than that of TFIIH/Ab-p44 containing p8 (bottom, NER, lanes 1 and 2). The addition of recombinant p8 strongly stimulates the repair activity of IIH9 up to the level of TFIIH/Ab-p44 (lanes 3 and 4). A similar increase of the specific DNA repair activity is obtained when the recombinant IIH10 complex, containing p8 (Figure 2B, top), is used (bottom, NER, compare lanes 5 and 6 with 1 and 2). In marked contrast, p8 is not required for TFIIH transcription activity (Figures 2A and 2B, bottom, Tx). This suggests a selective requirement of p8 for the NER versus the transcription activity of TFIIH.

Figure 1. p8 Is Part of a TFIIH Active in Repair and Transcription (A) TFIIH (Heparine Ultrogel [Gerard et al., 1991]) was immunoprecipitated with Ab-p44 and washed with increasing salt concentrations. TFIIH/Ab-p44 was eluted with a competitor peptide, and the samples were resolved by SDS-PAGE. The blot was silver stained (Staining) or immunostained (WB). The subunits of TFIIH are indicated. (B) TFIIH/Ab-p44 ([A], lane 2) was incubated with Ab-p8 antibody and eluted with a competitor peptide. The input (lane 1), the supernatant of the IP (lane 2), and the TFIIH/Ab-p8 (lane 3) were resolved by SDS-PAGE and immunostained (top). Fifty and 100 ng of the fractions were tested either in a dual incision (NER) or transcription assay (Tx). Sizes of the incision products or transcripts are indicated. (C) Bacterially expressed core TFIIH subunits (lanes 1–4) were tested for their ability to interact with GST-tagged p8 (lanes 6, 8, 10, and 12) or GST alone (lanes 5, 7, 9, and 11) coupled to glutathione-agarose beads. Proteins on the resin were resolved by SDS-PAGE and immunostained.

p8 and CAK Direct the TFIIH Repair and Transcription Activities We demonstrated above that p8 directly interacts with the XPD helicase, which was already shown to integrate CAK into TFIIH (Keriel et al., 2002). Furthermore, previous studies have shown that CAK stimulates transcription (Lu et al., 1992), whereas it is dispensable for NER in vitro (Araujo et al., 2000, Sung et al.,1996). Then, we asked whether p8 and CAK would compete or combine their action to direct the core TFIIH toward NER and/or transcription. Recombinant IIH6 (containing XPD and the five core TFIIH subunits XPB, p62, p52, p44, and p34) and IIH9 complexes were produced in insect cells (Figure 3A, left) and tested in an in vitro repair or transcription assay. Addition of increasing amounts of p8 to an incision assay containing IIH6 stimulates the

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Figure 2. p8 Specifically Engages TFIIH in Repair (A) Top, TFIIH/Ab-p44 (lane 1) or IIH9 (lane 2) was resolved by SDSPAGE and immunostained. Bottom, 100 ng of TFIIH/Ab-p44 (lane 1) or IIH9 (lanes 2–4) was tested in an NER or in a Tx in the presence of 1 and 3 ng of recombinant p8 (lanes 3 and 4). Sizes of the incision products or transcripts are indicated. (B) Top, IIH9 and IIH10 containing the nine (lane 1) or the ten (lane 2) subunits of TFIIH were purified and resolved by SDS-PAGE and immunostained. Bottom, 50 and 100 ng of IIH9 (lanes 1 and 2 and 3 and 4) or IIH10 (lanes 5 and 6) was tested in an NER or a Tx. Three nanograms of recombinant p8 was added to the reaction mixtures in lanes 3 and 4.

removal of the damaged oligonucleotide as efficiently as IIH9 does after addition of p8 (Figure 3A, right, NER, compare lanes 4–6 and 1–3). Addition of CAK to IIH6 allows optimal RNA synthesis (Figure 3A, lane 10) independently of p8 (lanes 7 and 8) and does not impair the stimulation of repair by p8 (compare lanes 6 with 7 and 8). Finally, p8 per se does not stimulate the transcription activity of the IIH6 and IIH9 complexes (compare lanes 4–6 and 1–3). Together, these data point to distinct roles for the two TFIIH components: p8 specifically triggers the repair activity of TFIIH, whereas CAK has a role in transcription. Importantly, both components act independently of each other, in a noncompetitive manner. To further examine whether p8 and CAK were part of TFIIH when it operates in transcription and repair, respectively, we designed the following challenge experiments. IIH9 was incubated together with the basal transcription factors, RNA Pol II, and the immobilized AdMLP template in the presence of ATP for 15 min at 30ºC with or without addition of p8 (Figure 3B, Incub 1). After extensive washing, the immobilized transcription complex was further incubated (Incub 2) with the Pt-DNA and repair factors without TFIIH. In those conditions, p8 is able to join the IIH9 complex on the AdMLP template (Incub 1) to further participate to the removal of the damaged oligonucleotide in the Pt-DNA template (Incub 2) (lanes 3–5). Conversely, IIH6 and p8 in the presence or absence of CAK were incubated together with XPC-hHR23B, XPA, RPA, XPG, and the biotinylated immobilized Pt-DNA in the presence of ATP 15 min at 30ºC (Figure 3C, Incub 1). After extensive washing, the dam-

aged DNA and the remaining associated repair factors were further incubated with AdMLP and the basal transcription factors without TFIIH (Incub 2). In those conditions, CAK associates with the NER factors assembled on the damaged DNA to further contribute to RNA synthesis in Incub 2 (lanes 3–5). In the absence of CAK in Incub 1, IIH6/p8 bound to the damaged DNA can move to the transcription template and allow RNA synthesis only when CAK is added together with the transcription factors in Incub 2 (lane 5). Finally, to assess the composition of the TFIIH complex that translocates to the sites of DNA photolesions in vivo, we locally UV irradiated FB789 primary human fibroblast nuclei (Volker et al., 2001). We previously showed colocalization of XPB and p8 at local damage sites (Giglia-Mari et al., 2004). In our experiment, enhanced fluorescence signals of XPB colocalize with CPD (Mori et al., 1991) within 30 min after local UV irradiation (Figure 3D, top panels). Additionally, cdk7 colocalizes with XPB in damage spots (bottom panels), indicating that CAK translocates to the UV-damaged DNA together with the core-TFIIH subunits. Altogether, these in vitro and in vivo findings suggest that the multifunctional TFIIH complex can interact with transcription and damage sites and fulfill its roles without major changes in its composition. p8 Stimulates the XPB ATPase Activity and Allows Damaged DNA Opening Next, we explored the function of p8 in NER. After addition of increasing amounts of p8 to IIH9, we first observed a proportional increase of the 50 and 30 incisions by the ERCC1-XPF and XPG endonucleases, respectively (Figure 4A, bottom), that parallels with the excision of damaged oligonucleotide (top). To elucidate whether p8 was involved in the opening of the DNA around the lesion, a prerequisite for the dual incision, we set up a permanganate footprinting assay (Tapias et al., 2004). Addition of p8 to a reaction mixture containing XPC-hHR23B and IIH9 allows extension of DNA opening as indicated by an increased sensitivity of nucleotides at positions T-4, T-5, and to a lesser extent, T-7 (Figure 4B, compare lanes 5 and 6), thereby mimicking the opening observed with TFIIH/Ab-p44 (lane 3). These data indicate that p8 stimulates the first steps of DNA opening around the lesion. Further addition of XPA to either TFIIH/Ab-p44 or IIH9 allows extension of the open DNA structure, as visualized by a marked sensitivity of T-7 and T-10 nucleotides (compare lanes 3 and 4 and lanes 6 and 7), only when p8 is present in the reaction mixture (lanes 7 and 8), suggesting that the extension of the opening triggered by XPA is p8 dependent. To unravel the molecular details of the involvement of p8 in NER, we tested its ability to modulate TFIIH enzymatic activities required for DNA damage opening. First, we investigated whether TFIIH ATPase activities, required for NER (Sung et al., 1996), would be regulated by p8. We observe that the IIH9 ATPase activity is increased after addition of p8, only in the presence of DNA (Figure 4C, compare lane 2 with lanes 4 and 9). A stimulation of the DNA-dependent IIH9 ATPase activity is also observed when XPC-hHR23B, which interacts with TFIIH (Yokoi et al., 2000), is added to the reaction mixture (compare lane 2 with lanes 3 and 7). Together,

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Figure 3. p8 and CAK Interplay in Transcription and Repair (A) Left, IIH6 and IIH9 containing the six subunits of the core without (lane 1, IIH6) or with (lane 2, IIH9) the subunits of the CAK complex were resolved by SDS-PAGE and immunostained. Right, 100 ng of IIH9 (lanes 1–3 and 9) or IIH6 (lanes 4–8 and 10) was incubated with p8 (1 and 3 ng) and/or CAK (50 and 100 ng) in an NER or a Tx assay. (B) Transcription initiation complex containing RNA Pol II, TFIIA, IIB, TBP, IIE, IIF, and when indicated, recombinant IIH9 and p8 on an immobilized AdML template was incubated 15 min at 30ºC with ATP (Incub 1). Beads were washed to remove unbound proteins and further incubated in a reconstituted dual incision assay containing XPC-hHR23B, XPA, RPA, XPG, XPF, and a Pt-DNA template (Incub 2). TFIIH/Ab-p44, lane 1, has been purified in Figure 1A. (C) Incision complex containing XPC-hHR23B, XPA, RPA, XPG and TFIIH/Ab-p44, IIH6, p8, and/or CAK as indicated was assembled on an immobilized Pt-DNA template and incubated 15 min at 30ºC with ATP (Incub 1). Beads were washed and further incubated in a reconstituted transcription assay containing RNA Pol II, TFIIA, IIB, TBP, IIE, IIF, and the AdMLP template (Incub 2). (D) Wt human primary fibroblasts (FB789) were UV irradiated with 70 J/m2 through a 3 mm pore filter and fixed 30 min later. Immunofluorescent labeling was performed with a rabbit polyclonal antibody, anti-XPB (panels a and e), a monoclonal mouse anti-CPD (panel b), or a monoclonal mouse anti-cdk7 (panel f). Nuclei were counterstained by DAPI, and slides were merged.

XPC-hHR23B and p8 combine their action to allow an optimal IIH9 ATPase activity (lanes 5, 8, and 10). To identify which ATPase activity was stimulated by p8, we took advantage of two IIH9 complexes mutated in either the XPB(K346R) or the XPD(K48R) ATP binding site (Tirode et al., 1999). We show that p8 stimulates the ATPase

activity of IIH9/XPD(K48R), but not the one of IIH9/ XPB(K346R) (lanes 12–15). We also observe that, within TFIIH, the residual XPD ATPase activity (lane 12) is much weaker than the residual XPB ATPase (lane 14), the last being stimulated by XPC-hHR23B. In a KMnO4 assay, each mutation in one of the ATP binding sites of either

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Figure 4. p8 Stimulates DNA Opening (A) One-hundred nanograms of IIH9 (lanes 3– 7) was tested either in the dual (top) or in the 50 (bottom, 77 nt) incisions of the Pt-DNA in the presence of 1–8 ng of p8 (lanes 4–7). Lane 1 contains TFIIH/Ab-p44. Lane 2 contains all the NER factors except TFIIH. The 30 incision being made first, 30 uncoupled incision by XPG (107 nt product) can be detected with the 50 labeled probe (Coin et al., 2004). (B) Pt-DNA was incubated with 10 ng of XPChHR32B (lanes 1 and 3–14) and 300 ng of either TFIIH/Ab-p44 (lanes 3 and 4), IIH9 (lanes 5–8), IIH9/XPB(K346R) (lanes 9–11), or IIH9/ XPD(K48R) (lanes 12–14). XPA (25 ng) and p8 (2 ng) were added when indicated. Lane 2, Pt-DNA with BSA only. Residues are numbered with the central thymine of the crosslinked GTG sequence designated T0. Arrows indicate KMnO4-sensitive sites. Adducted strand residues to the 30 and 50 of T0 are denoted by positive and negative integers (+N, 2N). (C) One-hundred nanograms of either IIH9 (lanes 2–9), IIH9/XPB(K346R) (lanes 12 and 13), or IIH9/XPD(K48R) (lanes 14 and 15) was tested in an ATPase assay in the presence of 50 ng of XPC-hHR23B, 2 ng of p8, and 120 ng of double-strand circular DNA as indicated at the top of the figure. Lanes 1 and 16 contain ATP only. A quantification of the inorganic phosphate (Pi) release and ATP was done by a Bio-imaging analyzer, and the ratio presented at the bottom of the figure shows the percentage of release of Pi.

XPB or XPD impairs the damaged DNA opening (Figure 4B, lanes 9–14). This suggests that the stimulation of the ATPase activity of XPB by p8 and XPC leads to the local opening of the DNA around the lesion. p8 Promotes the Translocation of XPA to UV Damage Sites The recruitment of the GG-NER factors to damaged DNA is initiated by XPC-hHR23B followed by the arrival of TFIIH and XPA, respectively (Riedl et al., 2003). We thus addressed the question whether the arrival of XPA to the lesion was dependent on p8 in vivo. We used the local UV irradiation technology combined with fluorescent antibody labeling in wild-type (wt) MRC5 and p8-defective TTD-A cells. Indeed, TFIIH from TTD1BRSV (TTD-A) cells is lacking p8 (Figure 5A). In vivo, enhanced fluorescence signals of XPB colocalize with

CPD spots within 30 min after local UV irradiation of both MRC5 (Figure 5B, panels a–d) and p8-defective TTD1BR-SV cells (panels i–l). By contrast, XPA colocalizes with XPB in MRC5 (panels e–h) but is absent in the locally UV-irradiated sites in TTD1BR-SV (panels m–p), underlying the crucial role of p8 in the recruitment of XPA in vivo. The arrival of XPA on the damaged DNA was further studied in vitro. We analyzed the composition of an intermediate NER complex assembled on damaged DNA by preincubating (Preinc) an immobilized biotinylated damaged DNA with XPC/hHR23B, IIH9, and XPA with or without p8 (Figure 5C). The immobilized DNA was subsequently washed to remove the nonspecifically bound proteins, and the remaining damaged DNA-associated protein fraction was analyzed by the omission of a single NER factor in a subsequent dual incision assay (Inc) (Riedl et al., 2003). The immobilized factors are able

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Figure 5. Recruitment of XPA at Sites of UV Damage (A) TFIIH from MRC5 or TTD1BR-SV total cell extracts was immunoprecipitated with Ab-p44 and eluted with a competitor peptide. The eluates were loaded on a 11% SDS-PAGE gel for immunostaining (WB) or on a 15% SDS-PAGE gel for silver staining (Staining). (B) MRC5 or TTD1BR-SV fibroblasts were UV irradiated as indicated in Figure 3D and labeled with a polyclonal anti-XPB (panels b and j), a monoclonal mouse anti-CPD (panels a, e, i, and m), or a polyclonal rabbit anti-XPA (panels f and n). (C) Immobilized damaged DNA was incubated with XPC/hHR23B, XPA, IIH9, and p8 as indicated (Preinc) for 15 min at 30ºC with ATP. DNA was further washed, supplemented with p8, RPA, ERCC1-XPF, and XPG, and subjected to dual incision assay in which one of the XPA, XPC, or IIH9 factors was omitted per lane as indicated (Inc). In lanes 1 and 5, no NER factor was added in the incubation. (D) XPC-HR23B, IIH9, and p8 were incubated with the Pt-DNA as indicated (Preinc) for 15 min at 30ºC with ATP. Beads were washed, and reactions were further supplemented with XPA, RPA, XPG, and ERCC1-XPF with or without IIH9 and p8 (Inc). Incision was allowed to continue for an additional 90 min.

to support incision when added to a reaction mixture containing all the repair factors except XPC/hHR23B and TFIIH (Inc), whether or not p8 is added in the Preinc (Figure 5C, lanes 3 and 4 and 7 and 8). In marked contrast, XPA is unable to bind the damaged DNA in the absence of p8 in the Preinc (compare lanes 2 and 6). To determine the minimum set of factors required for the binding of p8 to the repair complex, XPC-hHR23B/PtDNA or XPC-hHR23B/IIH9/Pt-DNA was preassembled in the presence or absence of p8. After washes, the resulting complexes were analyzed in a dual incision assay supplemented by XPA, RPA, XPG, ERCC1/XPF with or without IIH9, and p8. We found that p8 does not bind to the XPC/hHR23B-damaged DNA complex by itself (Figure 5D, lane 3) unless IIH9 is present (lanes 6 and 7). Altogether, these results demonstrate that the arrivals of XPC and TFIIH to the site of DNA damage are not dependent on p8. However, XPA accumulates in these sites only when p8, within TFIIH, is recruited to the damage site already recognized by XPC-hHR23B.

The p8 N Terminus Is Crucial for Optimal XPB ATPase Activity and NER The above findings prompted us to identify which residues of this polypeptide were important for NER. We then produced several recombinant N- and C-terminaltruncated p8 proteins (Figure 6A, top). When added to a reconstituted incision assay, p8CD15 lacking the last C-terminal 15 amino acids allows the incision of the damaged oligonucleotide (Figure 6A, lanes 9 and 10), whereas p8ND10 and p8ND20 N-terminal-truncated polypeptides are not able to induce the removal of the lesion (compare lanes 5–8 with 3 and 4). Next, we injected the constructs expressing either wt or ND10 p8 in TTD1BR, and we evaluated their ability to restore UV-induced DNA repair synthesis (UDS) after UV irradiation and incubation with radioactive thymidine (Stefanini et al., 1993). The repair defect in TTD1BR fibroblasts is restored after microinjection of the wt p8, as clearly demonstrated by the drastic increase in the radioactive grain number per nuclei (Figure 6B).

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Figure 6. Analysis of N- and C-Terminal Deletions of p8 (A) Top, schematic representation of the different p8 constructs. Bottom, IIH9 (100 ng) was tested in a dual incision assay with XPC-hHR23B, XPA, RPA, XPG, ERCC1-XPF, and Pt-DNA in the presence of 1 and 3 ng of p8 (lanes 3 and 4), p8ND10 (lanes 5 and 6), p8ND20 (lanes 7 and 8), and p8CD15 (lanes 9 and 10). (B) Plasmids expressing p8 or p8ND10 were microinjected in TTD1BR fibroblasts. UDS (number of radioactive grains in the nucleus) is measured after UV irradiation and incubation with radioactive thymidine and expressed as grains/nucleus (mean of >50 nuclei/sample 6 SEM). (C) A KMno4 assay, as described in Figure 4B, was performed with 300 ng of either TFIIH/Ab-p44 (lane 2) or IIH9 (lanes 3–5) in the presence of 3 and 6 ng of either p8 (lanes 4 and 5) or p8ND10 (lanes 6 and 7). (D) One-hundred nanograms of TFIIH/Ab-p44 (lane 3) or IIH9 (lanes 2 and 4–7) was assayed in an ATPase assay in the presence of 50 ng of XPChHR23B and 120 ng of double-strand circular DNA in each lanes as well as 1 and 2 ng of either p8 (lanes 4 and 5) or p8ND10 (lanes 6 and 7). In lanes 8 and 9, 2 ng of p8 and p8ND10 was tested as control. A quantification of the Pi release was performed as described in Figure 4C.

In contrast, microinjection of p8ND10 is unable to correct the repair defect in TTD-A cells (Figure 6B). Furthermore, microinjection of p8ND10 in wt primary fibroblasts induces a drop of 40% of the UDS, indicating a dominant-negative effect of this mutant on NER in vivo (see the Supplemental Data available with this article online). KMnO4 footprinting and ATPase assays further demonstrated that the deletion of the first ten residues of p8 prevents the opening of the DNA (Figure 6C, lanes 4–7) and strongly impairs the stimulation of the ATPase activity of XPB (Figure 6D, compare lanes 4 and 5 with 6 and 7). These data demonstrate the importance of the first ten N-terminal residues of p8 in the stimulation of XPB ATPase activity and the role of such stimulation in the repair activity of TFIIH. p8 Restores the TFIIH Cellular Concentration in XPD/TTD Cell Lines Mutations in p8 are associated with a reduced cellular amount of TFIIH in TTD-A cells (Giglia-Mari et al.,

2004), suggesting an additional role of p8 in the regulation of the steady-state level of TFIIH. To gain insight into this additional role of p8, MRC5 fibroblasts were labeled with latex blue fluorescent beads, transiently transfected with an expression vector containing an antisense RNA directed toward p8 messenger RNA and analyzed for TFIIH levels by immunofluorescence. Using confocal microscopy and antibodies toward the XPB subunit of TFIIH, we found that the intensity of the XPB staining in the nucleus is reduced 60 hr after transfection in MRC5 (compared to untransfected MRC5) and is similar to that observed in TTD1BR-SV cells (Figure 7A), whereas TBP (the TATA-box binding protein) staining does not reveal significant differences (data not shown). Not only p8 mutations but also all the mutations in XPD found in TTD patients cause a substantial reduction of the level of TFIIH (Botta et al., 2002). Because XPD and p8 interact with each other, we were wondering whether the reduction of TFIIH in TTD cells mutated in XPD (TTD-XPD) could be restored by overexpressing

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Figure 7. p8 Stabilizes the TFIIH Complex (A) Expression vector encoding an antisense RNA toward p8 mRNA was transfected in MRC5 fibroblasts, previously labeled by latex blue fluorescent beads. The TFIIH level was measured 60 hr after transfection, using antibodies directed toward the XPB subunit (right) in either untransfected MRC5 (MRC5), transfected MRC5 (MRC5 p82/2), or TTD-A (TTD1BR-SV) cells spotted on the same slide. Nuclei were counterstained by DAPI, and slides were merged (left). (B) Expression vector encoding both p8 and green fluorescent protein (GFP) was either transfected (panels a, b, e, f, I, and j) or microinjected (panels d, h, and l) into TTD1BR (top panels), TTD8PV (TTD-XPD) (middle panels), and TTD1BEL (TTD-XPD) (bottom panels) cells. The level of TFIIH was measured in transfected cells by using Ab-XPB, and the level of repair was measured on cells after incubation with [3H]thymidine and autoradiography. Graphs show the amount of XPB (gray) or TBP (white) on the various transfected or untransfected cells as measured by prism spectrophotometer system. Nuclei were counterstained by DAPI, and slides showing XPB, GFP, and DAPI were merged.

p8. TTD1BR (p8-L21P/R56stop), TTD8PV (XPD-R112H/ R112H), or TTD1BEL (R722W/R616P) fibroblasts were transfected with the pIRES-EGFP/p8 bicistronic expression vector, which coexpresses both the green fluorescent protein GFP and p8, and analyzed by confocal microscopy using Ab-XPB antibodies. The intensity of the XPB staining is reduced in all noninjected cells (Figure 7B panels a/b, e/f, and i/j), whereas TBP (the TATA-box binding protein) staining does not reveal significant differences (panels c, g, and k). The transfection of p8 restores the TFIIH concentration not only in the

TTD1BR cells that carry the p8 mutations (panels a/b) but also in both XP-D cell strains, TTD8PV and TTD1BEL (panels e/f and i/j; see also histograms panels g and k). The restoration of the cellular TFIIH concentration is gradual, with a maximum reached 60 hr after transfection (panel c). The transfection of a vector expressing the p44 subunit of TFIIH, another partner of XPD (Coin et al., 1998), has no effect (data not shown) on the TFIIH level. Interestingly, microinjection of p8 in TTD8PV cells (homozygotes for the R112H mutation) also results in a consistent increase of UV-induced DNA repair synthesis

p8/TTDA and TFIIH Repair Activity 223

Table 1. Microinjection of p8 cDNA into TTD Fibroblasts UV-Induced DNA Synthesisa Cell Strain

Mutated Gene

AA Changes

Noninjected

Injected with p8

TTD1BR TTD8PV TTD1BEL

p8 XPD XPD

R56stop/L21P R112H/R112H R722W/R616P

13.9 6 0.8 12.9 6 0.4 13.1 6 0.7

34.5 6 2.1 19.8 6 1.9 13.8 6 0.9

a

UDS, measured as the radioactive thymidine incorporation, is expressed as grains/nucleus 6 SEM (>50 nuclei/sample).

(UDS) similar to what is observed in TTD1BR (Table 1 and Figure 7B, panels d and h, compare the microinjected cell in the bottom panels with the noninjected one in the top panels). By contrast, overexpression of p8 does not restore repair in TTD1BEL cells bearing the XPD-R722W and R616P mutations (Table 1 and Figure 7B, panel l). These data support the notion that p8 is a key regulator of the intracellular level of TFIIH. An overexpression of p8 is able to correct the decrease in the concentration of TFIIH, a common phenotype of TTD cells and also, in some cases, to correct the UDS. Discussion We demonstrated here how p8/TTD-A orchestrates the TFIIH repair activity but is dispensable for transcription. Our work reveals a specialization of TFIIH components to direct the complex toward repair or transcription. Pinpointing the Role of p8 in Repair An established view of the GG-NER pathway is that TFIIH targets the damaged DNA already recognized by XPC-hHR23B (Riedl et al., 2003; Volker et al., 2001). Thereafter, ATP hydrolysis by TFIIH is expected to promote the unwinding of the DNA and the relocation of the XPC factor (Tapias et al., 2004). The recruitment of XPA, RPA, XPG, and ERCC1/XPF to the preincision complex may then occur. Of interest was to investigate how p8 contributes to the removal of the lesion. We demonstrated that p8 functions in repair as an integral part of TFIIH. Furthermore, we showed that p8 stimulates the early unwinding/opening of the DNA by the XPB and XPD helicases and the late 30 and 50 incisions by XPG and XPF nucleases. In vivo, we observed that the recruitment of TFIIH to UV-induced DNA damages was effective in TTD-A cells and the arrival of XPA, the third factor that targets damaged DNA in NER, was impaired, establishing an early role of p8 in the NER reaction. KMnO4 footprinting details the role of p8 on NER where it initiates the damaged DNA opening further enlarged and stabilized by the arrival of XPA. We indeed found that p8 stimulates the ATPase activity of XPB in a DNA-dependent manner and demonstrated that such ATPase activity depends not only on DNA but also on XPC-hHR23B, a factor that strongly interacts with TFIIH (Yokoi et al., 2000). Moreover, the stimulation of the ATPase activity of XPB by p8 is not accompanied by an increase of its helicase activity (data not shown), suggesting that these activities may not be entirely connected. Using either GST-pull down assay or coexpressions in baculovirus-infected cell, we were not able to find any interaction between p8 and XPB. However, they both in-

teract with a common partner, p52 (Jawhari et al. [2002] and this work), that may act as an intermediate in the stimulation of XPB ATPase by p8. Finally, the deletion of the first ten residues of p8 impairs the stimulation of the XPB ATPase and leads to loss of NER activity, implying that such stimulation is crucial for the TFIIH repair activity. Altogether, our results support a scenario in which the recruitment of TFIIH to the damaged DNA, primarily recognized by XPC, occurs without the help of p8 (the present work) and in an ATP-independent manner (Riedl et al., 2003). The contact of TFIIH with the XPChHR23B/damaged DNA complex might then condition a conformational change of the preincision complex catalyzed by p8, in the presence of ATP (Tapias et al., 2004). XPB, with the help of p8, would act as an ATP-driven motor that supplies the required energy to reorganize the intermediate DNA repair complex, thereby supporting the repositioning of XPC-hR23B, the unwinding of DNA by XPD, and the recruitment of XPA. In our model, the XPB ATPase activity supports the remodeling of the preincision complex before the arrival of XPG and ERCC1/XPF endonucleases and then is not only restricted to the role of supplier of energy to its helicase moiety. Furthermore, this hypothesis allows the role of XPB in NER to be brought into harmony with its role in promoter opening during RNA Pol II-dependent transcription. Indeed, the XPB ATPase has been postulated to remodel the RNA Pol II preinitiation complex to allow elongation and the setting up of the various RNA processing steps (Lin et al., 2005). Conversely, the XPD helicase activity is necessary for NER where it would allow for DNA damage opening but have a minor role in transcription (Guzder et al., 1994a; Tirode et al., 1999). Such a model in which XPB ATPase is involved in the remodeling of the protein/DNA complexes while XPD is directly involved in damage DNA opening also explains why a mutation in the ATP binding site of either XPB or XPD totally abolishes the formation of the open DNA structure during NER in a KMno4 assay. XPD, a ‘‘Seat’’ for p8 and CAK The most striking feature of TFIIH is its multifunctionality, particularly its versatile engagement in completely distinct processes. The finding that both p8 and CAK target XPD and the specific role of p8 in DNA repair raised the question of a possible competition/connection between these components. For instance, mutation in one of them might disturb the function of the other one. Indeed, some XPD mutations prevent not only DNA repair but also transcription activation regulated by CAK, thereby explaining at least partially the

Molecular Cell 224

deficiency in hormonal responses in XP/TTD patients (Chen et al., 2000; Keriel et al., 2002). We demonstrated that p8 and CAK, as parts of a same TFIIH complex working in repair and transcription (Hoogstraten et al., 2002), act independently of each other to regulate distinct TFIIH activities. p8 allows optimal removal of damaged oligonucleotides when associated with either IIH6 or IIH9, indicating that the presence of CAK was not required for the opening/incision/ excision steps of NER (Sung et al., 1996; Araujo et al., 2000). However, local UV irradiation experiments on primary fibroblasts clearly demonstrate the presence of CAK on the repair complex 30 min after irradiation, suggesting that the CAK complex is translocated with the core-TFIIH to damaged sites. Then one cannot exclude that CAK, bound to lesions through its interaction with the core-TFIIH, plays a role in the very early steps of the cell response to DNA damages (i.e., chromatin remodeling or cell signaling). Conversely, in vitro RNA synthesis is CAK dependent and it does not require p8. Such data support the previous observations showing that basal transcription was not affected in TTD-A cells (Vermeulen et al., 2000) and are suitable with the observation that absence of p8 in TTD-A cells is compatible with life (Giglia Mari et al., 2004). In agreement with these data, we also demonstrated that the transactivation of a reporter gene under the control of nuclear receptors was not affected in TTD-A cells in which p8 is absent (Keriel et al., 2002). However, we cannot exclude that the transcription of specific genes expressed at very high levels might be affected by the lowered TFIIH concentration or by mutations in p8, explaining how defects in p8 would contribute to transcriptional defects. Altogether, our data offer insight into the specialization of some of the TFIIH components. Some subunits are shared between transcription and DNA repair (i.e., XPB and XPD), whereas others are more specifically oriented (i.e., p8 is required for DNA repair and CAK for transcription). p8 Is a Key Factor Regulating TFIIH Stabilization TTD is associated with mutations in the three subunits of TFIIH: XPB, XPD, or p8 (Lehmann, 2003). Mutations in any of the three genes cause a decrease by up to 70% in the cellular concentration of TFIIH (Botta et al., 2002). One obvious mechanism, explaining the cellular phenotype of TTD, is that a mutation affecting the stability of one subunit renders the entire complex unstable. By using antisense RNA, we demonstrated that the silencing of p8 expression reduces the TFIIH intracellular concentration. Conversely, overexpression of p8 is able to restore a normal level of TFIIH not only in TTD-A cells but also in TTD cells mutated in the XPD gene, the most common defect within NER-defective TTD patients (Giglia-Mari et al. [2004] and this paper). In contrast, overexpression of XPD in TTD-A cells has no effect on the TFIIH level (Vermeulen et al., 2000). These results defined p8 as a key regulator of the TFIIH cellular level, probably either by providing stability to the TFIIH complex or by protecting it from proteolytic degradation. Interestingly, overexpression of p8 protein increased UDS in cell lines bearing mutations in the Nterminal region of XPD. In marked contrast, when both alleles of XPD are mutated in the C-terminal region,

overexpression of p8 is not accompanied by a an increased UDS, suggesting that p8 fails to restore the accurate XPD/p44 interaction that allows a maximal XPD helicase activity (Coin et al., 1998). Altogether, our data demonstrate that p8 combines two functions in TFIIH by regulating its cellular concentration and its DNA repair activity. Consequently, this work increases the potential diversity of therapies that can be performed to treat the peculiar clinical symptoms of the trichothiodystrophy disorder. Experimental Procedures Cell Lines and Culture We cultured primary the human fibroblasts TTD8PV, TTD1BEL, TTD1BR, and FB789 (wt, NER proficient) under standard conditions at 37ºC, 5% CO2, and 3% oxygen in Ham’s F-10 medium supplemented with 12% fetal calf serum and antibiotics (penicillin and streptomycin). We cultured the transformed cell lines TTD1BR-SV (TTD-A) and MRC5 (wt) at 37ºC, 5% CO2 in a 1:1 mixture of Ham’s F-10 medium and Dulbecco’s modified Eagle medium (Gibco) supplemented with 8% fetal calf serum and antibiotics. Construction of Plasmids Baculoviruses overexpressing p8 were constructed in pSK278 vector. p8 was inserted at the BamHI/EcoRI site, in fusion with the FLAG tag in the N-terminal end of p8. The deletion of the first 10, 20, and last 15 amino acids of p8 were obtained by site-directed mutagenesis (QuikChange, Stratagene). The resulting vectors were recombined with baculovirus DNA (BaculoGold DNA, PharMingen) in Sf9 cells. For GST-pull down assay, p8 was inserted into pGEX-3T (Pharmacia) and the subunits of the core TFIIH into pET-15b (Novagen) expression vectors. For immunofluorescence analysis, p8 was inserted into the pIRES2-EGFP expression vector (Clontech). For in vivo repair assay, p8 (wt or mutant) was inserted into the pcDNA3(+) expression vector (Invitrogen). A gene encoding an antisense RNA molecule was obtained by inverting the orientation of the cDNA of p8 in the pcDNA3(+) plasmid expression vector. Protein Purification Recombinant wt or mutated TFIIH and CAK complexes were purified as in Tirode et al. (1999). The p8 protein was purified with the antiFLAG M2 antibody agarose affinity gel (Sigma-Aldrich). TFIIH from HeLa was immunoprecipitated with Ab-p44 or Ab-p8 antibodies linked to protein A Sepharose and further eluted with a competitor peptide (Coin et al., 1999). GST Pull Down GST or GST-p8 polypeptides were expressed in E. coli and incubated with glutathione-agarose beads. The various subunits of the core TFIIH were expressed in E. coli (1 3 106 cells), and cell lysates were incubated with 5 mg of GST or GST-p8 proteins bound to beads at 4ºC for 1 hr. Pull downs were analyzed by Western blotting. ATPase Assay Protein fractions were incubated for 2 hr at 30ºC in the presence of 1 mCi [g-32P]ATP (7000 Ci/mmol, ICN Pharmaceuticals) in a 20 ml reaction volume in 20 mM Tris-HCl (pH 7.9), 4 mM MgCl2, 1 mM DTT, 50 mg/ml BSA, and when indicated, 120 ng of supercoiled doublestrand DNA (pSK). Reactions were stopped by adding EDTA to 50 mM and SDS to 1% (w/w). The reactions were then diluted 5-fold, spotted onto polyethylenimine (PEI) TLC plates (Merck), run in 0.5 M LiCl/1 M formic acid, and autoradiographed. Protein Binding Studies on Immobilized DNA Dynabeads M-280 Streptavidin (Dynal) coupled to DNA were incubated in 10 mM HEPES, 100 mM glutamate, 10 mM MgOAc, 5 mM EGTA, 3,5% glycerol, 60 mg/ml casein, 5 mg/ml PVP, and 2.5 mM DTT 15 min at room temperature to limit unspecific binding of proteins (Ranish et al., 2004). Immobilized DNA was then incubated with a reconstituted dual incision assay containing recombinant XPC-hR23B, XPA, RPA, TFIIH, and XPG at 30ºC, 15 min with 2 mM

p8/TTDA and TFIIH Repair Activity 225

ATP. Upon incubation, magnetic beads were collected on a magnetic particle concentrator (Dynal MPC) and supernatants removed. Beads were then washed five times in four volumes of cold incision buffer and resuspended in the same buffer for functional analysis of bound fractions by single NER factor omission in a subsequent dual incision assay (Riedl et al., 2003). Local UV Irradiation The cells were rinsed with PBS and were covered with an isopore polycarbonate filter with pores of 3 mm diameter (Millipore, Badford, MA). Cells were then exposed to UV irradiation with a Philips TUV lamp (predominantly 254 nm) at a dose of 70 J/m2 (Volker et al., 2001). Subsequently, the filter was removed, the medium was added back to the cells, and cells were returned to culture conditions for 30 min. Fluorescence MRC5 fibroblasts were grown for 2 days with fluorescent latex beads (Fluoresbrite Carboxylate Microspheres, Polysciences). Cells were fixed in 3% paraformaldehyde for 10 min at room temperature and permeabilized with PBS/0.5% Triton for 5 min. After washing with PBS-Tween (0.05%), the slides were incubated for 1 hr with the indicated antibodies. After extensive washing with PBS-Tween, they were incubated for 1 hr with Cy3-conjugated goat anti-rabbit IgG (Jackson Laboratories) or with anti-mouse Alexa 488 IgG (Jackson Laboratories) diluted 1:400 in PBS-Tween/0.5% NFDM. The slides were counterstained for DNA with DAPI prepared in Vectashield mounting medium (Vector lab). All images were collected by a Leica Confocal TCS 4D microscope equipped with both UV laser and an Argon/Kripton laser, and standard filters to allow collection of the data at 488 and 568 nm. The software TCSTK was used for three-color reconstructions, and figures were generated by using the PLCHTK software. Microneedle Injection Nuclear microinjection of cDNA was performed according to a method described elsewhere (Stefanini et al., 1993). For each experiment, at least 150 cells were injected. Briefly, the cells were irradiated with a UV dose of 20 J/m2, incubated in medium containing 10 mCi/ml [3H]thymidine (3H-TdR, PerkinElmer Life and Analytical Sciences, Boston MA 02118 USA, specific activity 25 Ci/mmol), and processed for autoradiography 2 hr later. UDS was measured by counting the number of grains on the nucleus of at least 100 microinjected cells. Antibodies A monoclonal mouse antibody (Ab-p8) was raised against the polypeptide VGELMDQNAFSLTQK corresponding to residues 57–71 of p8. Primary antibodies (the final dilutions are indicated in parentheses) used in fluorescent labeling were rabbit IgG polyclonal anti-XPB (S-19, Santa Cruz Biotechnology) (1:200), rabbit IgG polyclonal antiXPA (1:200) (FL-273, Santa Cruz Biotechnology), mouse IgG monoclonal anti-CPD (TDM2) (1:2000) (MBL international corp.), and mouse IgG monoclonal anti-cdk7 (2F8) (Coin et al., 2004). Secondary antibodies used in this study were Alexa 488 anti-mouse IgG and Cy3-conjugated goat anti-rabbit IgG. Supplemental Data Supplemental Data include one figure and can be found with this article online at http://www.molecule.org/cgi/content/full/21/2/215/ DC1/. Acknowledgments We thank J.H. Hoeijmakers, W. Vermeulen, and G. Giglia-Mari for fruitful discussions. We are grateful to A. Larnicol for excellent technical expertise and to I. Kolb, J.L. Weickert, and the IGBMC services for their invaluable assistance. We are grateful to M. Demeny and Z. Nagy for critically reading the manuscript. This study was supported by grants from La Ligue contre le Cancer (e´quipe labelise´e, number EL2004), from the Action concerte´e incitative of the Ministe´re de l’Education Nationale et de la Recherche (BCMS, number 03 2 535), and the Institut des Maladies Rares (number A03098MS). O.Z. is supported by a Marie Curie Research Training Network Con-

tract (number MRTN-CT-2003-503618). M.S. is supported by the Fondazione Cariplo and Associazione Italiana per la Ricerca sul Cancro. L.P.D. is supported by the foundation pour la recherche me´dicale. Received: August 2, 2005 Revised: October 12, 2005 Accepted: October 25, 2005 Published: January 19, 2006 References Akoulitchev, S., and Reinberg, D. (1998). The molecular mechanism of mitotic inhibition of TFIIH is mediated by phosphorylation of CDK7. Genes Dev. 12, 3541–3550. Araujo, S.J., Tirode, F., Coin, F., Pospiech, H., Syvaoja, J.E., Stucki, M., Hubscher, U., Egly, J.M., and Wood, R.D. (2000). Nucleotide excision repair of DNA with recombinant human proteins: definition of the minimal set of factors, active forms of TFIIH, and modulation by CAK. Genes Dev. 14, 349–359. Botta, E., Nardo, T., Lehmann, A.R., Egly, J.M., Pedrini, A.M., and Stefanini, M. (2002). Reduced level of the repair/transcription factor TFIIH in trichothiodystrophy. Hum. Mol. Genet. 11, 2919–2928. Chen, D., Riedl, T., Washbrook, E., Pace, P.E., Coombes, R.C., Egly, J.M., and Ali, S. (2000). Activation of estrogen receptor alpha by S118 phosphorylation involves a ligand-dependent interaction with TFIIH and participation of CDK7. Mol. Cell 6, 127–137. Coin, F., Marinoni, J.C., Rodolfo, C., Fribourg, S., Pedrini, A.M., and Egly, J.M. (1998). Mutations in the XPD helicase gene result in XP and TTD phenotypes, preventing interaction between XPD and the p44 subunit of TFIIH. Nat. Genet. 20, 184–188. Coin, F., Bergmann, E., Tremeau-Bravard, A., and Egly, J.M. (1999). Mutations in XPB and XPD helicases found in xeroderma pigmentosum patients impair the transcription function of TFIIH. EMBO J. 18, 1357–1366. Coin, F., Auriol, J., Tapias, A., Clivio, P., Vermeulen, W., and Egly, J.M. (2004). Phosphorylation of XPB helicase regulates TFIIH nucleotide excision repair activity. EMBO J. 23, 4835–4846. Evans, E., Moggs, J.G., Hwang, J.R., Egly, J.M., and Wood, R.D. (1997). Mechanism of open complex and dual incision formation by human nucleotide excision repair factors. EMBO J. 16, 6559– 6573. Frit, P., Bergmann, E., and Egly, J.M. (1999). Transcription factor IIH: a key player in the cellular response to DNA damage. Biochimie 81, 27–38. Gerard, M., Fischer, L., Moncollin, V., Chipoulet, J.M., Chambon, P., and Egly, J.M. (1991). Purification and interaction properties of the human RNA polymerase B(II) general transcription factor BTF2. J. Biol. Chem. 266, 20940–20945. Giglia-Mari, G., Coin, F., Ranish, J.A., Hoogstraten, D., Theil, A., Wijgers, N., Jaspers, N.G., Raams, A., Argentini, M., van der Spek, P.J., et al. (2004). A new, tenth subunit of TFIIH is responsible for the DNA repair syndrome trichothiodystrophy group A. Nat. Genet. 36, 714–719. Guzder, S.N., Qiu, H., Sommers, C.H., Sung, P., Prakash, L., and Prakash, S. (1994a). DNA repair gene RAD3 of S. cerevisiae is essential for transcription by RNA polymerase II. Nature 367, 91–94. Guzder, S.N., Sung, P., Bailly, V., Prakash, L., and Prakash, S. (1994b). RAD25 is a DNA helicase required for DNA repair and RNA polymerase II transcription. Nature 369, 578–581. Hanawalt, P. (2002). Subpathways of nucleotide excision repair and their regulation. Oncogene 21, 8949–8956. Holstege, F.C., van der Vliet, P.C., and Timmers, H.T. (1996). Opening of an RNA polymerase II promoter occurs in two distinct steps and requires the basal transcription factors IIE and IIH. EMBO J. 15, 1666–1677. Hoogstraten, D., Nigg, A.L., Heath, H., Mullenders, L.H., van Driel, R., Hoeijmakers, J.H., Vermeulen, W., and Houtsmuller, A.B. (2002). Rapid switching of TFIIH between RNA polymerase I and II transcription and DNA repair in vivo. Mol. Cell 10, 1163–1174.

Molecular Cell 226

Itin, P.H., Sarasin, A., and Pittelkow, M.R. (2001). Trichothiodystrophy: update on the sulfur-deficient brittle hair syndromes. J. Am. Acad. Dermatol. 44, 891–920; quiz 921–924. Jawhari, A., Laine, J.P., Dubaele, S., Lamour, V., Poterszman, A., Coin, F., Moras, D., and Egly, J.M. (2002). p52 Mediates XPB function within the transcription/repair factor TFIIH. J. Biol. Chem. 277, 31761–31767. Keriel, A., Stary, A., Sarasin, A., Rochette-Egly, C., and Egly, J.M. (2002). XPD mutations prevent TFIIH-dependent transactivation by nuclear receptors and phosphorylation of RARalpha. Cell 109, 125–135. Lehmann, A.R. (2003). DNA repair-deficient diseases, xeroderma pigmentosum, Cockayne syndrome and trichothiodystrophy. Biochimie 85, 1101–1111. Lin, Y.C., Choi, W.S., and Gralla, J.D. (2005). TFIIH XPB mutants suggest a unified bacterial-like mechanism for promoter opening but not escape. Nat. Struct. Mol. Biol. 12, 603–607. Lu, H., Zawel, L., Fisher, L., Egly, J.M., and Reinberg, D. (1992). Human general transcription factor IIH phosphorylates the C-terminal domain of RNA polymerase II. Nature 358, 641–645. Mori, T., Nakane, M., Hattori, T., Matsunaga, T., Ihara, M., and Nikaido, O. (1991). Simultaneous establishment of monoclonal antibodies specific for either cyclobutane pyrimidine dimer or (6-4) photoproduct from the same mouse immunized with ultraviolet-irradiated DNA. Photochem. Photobiol. 54, 225–232. Ranish, J.A., Hahn, S., Lu, Y., Yi, E.C., Li, X.J., Eng, J., and Aebersold, R. (2004). Identification of TFB5, a new component of general transcription and DNA repair factor IIH. Nat. Genet. 36, 707–713. Riedl, T., Hanaoka, F., and Egly, J.M. (2003). The comings and goings of nucleotide excision repair factors on damaged DNA. EMBO J. 22, 5293–5303. Rochette-Egly, C., Adam, S., Rossignol, M., Egly, J.M., and Chambon, P. (1997). Stimulation of RAR alpha activation function AF-1 through binding to the general transcription factor TFIIH and phosphorylation by CDK7. Cell 90, 97–107. Schaeffer, L., Roy, R., Humbert, S., Moncollin, V., Vermeulen, W., Hoeijmakers, J.H., Chambon, P., and Egly, J.M. (1993). DNA repair helicase: a component of BTF2 (TFIIH) basic transcription factor. Science 260, 58–63. Schwartz, B.E., Larochelle, S., Suter, B., and Lis, J.T. (2003). Cdk7 is required for full activation of Drosophila heat shock genes and RNA polymerase II phosphorylation in vivo. Mol. Cell. Biol. 23, 6876–6886. Stefanini, M., Vermeulen, W., Weeda, G., Giliani, S., Nardo, T., Mezzina, M., Sarasin, A., Harper, J.I., Arlett, C.F., Hoeijmakers, J.H., et al. (1993). A new nucleotide-excision-repair gene associated with the disorder trichothiodystrophy. Am. J. Hum. Genet. 53, 817–821. Sugasawa, K., Ng, J., Masutani, C., Iwai, S., van der Spek, P., Eker, A., Hanaoka, F., Bootsma, D., and Hoeijmakers, J. (1998). Xeroderma pigmentosum group C protein complex is the initiator of global genome nucleotide excision repair. Mol. Cell 2, 223–232. Sung, P., Guzder, S.N., Prakash, L., and Prakash, S. (1996). Reconstitution of TFIIH and requirement of its DNA helicase subunits, Rad3 and Rad25, in the incision step of nucleotide excision repair. J. Biol. Chem. 271, 10821–10826. Takagi, Y., Masuda, C.A., Chang, W.H., Komori, H., Wang, D., Hunter, T., Joazeiro, C.A., and Kornberg, R.D. (2005). Ubiquitin ligase activity of TFIIH and the transcriptional response to DNA damage. Mol. Cell 18, 237–243. Tapias, A., Auriol, J., Forget, D., Enzlin, J.H., Scharer, O.D., Coin, F., Coulombe, B., and Egly, J.M. (2004). Ordered conformational changes in damaged DNA induced by nucleotide excision repair factors. J. Biol. Chem. 279, 19074–19083. Tirode, F., Busso, D., Coin, F., and Egly, J.M. (1999). Reconstitution of the transcription factor TFIIH: assignment of functions for the three enzymatic subunits, XPB, XPD, and cdk7. Mol. Cell 3, 87–95. Vermeulen, W., Bergmann, E., Auriol, J., Rademakers, S., Frit, P., Appeldoorn, E., Hoeijmakers, J.H., and Egly, J.M. (2000). Sublimiting concentration of TFIIH transcription/DNA repair factor causes TTD-A trichothiodystrophy disorder. Nat. Genet. 26, 307–313.

Volker, M., Mone, M.J., Karmakar, P., van Hoffen, A., Schul, W., Vermeulen, W., Hoeijmakers, J.H., van Driel, R., van Zeeland, A.A., and Mullenders, L.H. (2001). Sequential assembly of the nucleotide excision repair factors in vivo. Mol. Cell 8, 213–224. Yokoi, M., Masutani, C., Maekawa, T., Sugasawa, K., Ohkuma, Y., and Hanaoka, F. (2000). The xeroderma pigmentosum group C protein complex XPC-HR23B plays an important role in the recruitment of transcription factor IIH to damaged DNA. J. Biol. Chem. 275, 9870–9875. Zurita, M., and Merino, C. (2003). The transcriptional complexity of the TFIIH complex. Trends Genet. 19, 578–584.