ARCH domain of XPD, an anchoring platform for CAK that conditions TFIIH DNA repair and transcription activities

Abstract

The xeroderma pigmentosum group D (XPD) helicase is a subunit of transcription/DNA repair factor, transcription factor II H (TFIIH) that catalyzes the unwinding of a damaged DNA duplex during nucleotide excision repair. Apart from two canonical helicase domains, XPD is composed of a 4Fe-S cluster domain involved in DNA damage recognition and a module of uncharacterized function termed the "ARCH domain." By investigating the consequences of a mutation found in a patient with trichothiodystrophy, we show that the ARCH domain is critical for the recruitment of the cyclin-dependent kinase (CDK)-activating kinase (CAK) complex. Indeed, this mutation not only affects the interaction with the MAT1 CAK subunit, thereby decreasing the in vitro basal transcription activity of TFIIH itself and impeding the efficient recruitment of the transcription machinery on the promoter of an activated gene, but also impairs the DNA unwinding activity of XPD and the nucleotide excision repair activity of TFIIH. We further demonstrate the role of CAK in downregulating the XPD helicase activity within TFIIH. Taken together, our results identify the ARCH domain of XPD as a platform for the recruitment of CAK and as a potential molecular switch that might control TFIIH composition and play a key role in the conversion of TFIIH from a factor active in transcription to a factor involved in DNA repair.

Fig. 1.

TTD and XP mutations in the XPD TFIIH subunit. (A) Schematic representation of XPD. Helicase motor domains HD1 and HD2 are shown in magenta and blue, respectively, the Fe-S iron sulfur-containing domain is in cyan, and the ARCH domain is in green. Black bars indicate the helicase motifs (I, Ia, II, II, IV, V, and VI). Positions of mutations flanking the ARCH domain and diseases associated with the mutations are shown. (B) Production of rIIH. The 10 subunits of human TFIIH, including either wild-type or mutated XPD, were coexpressed in insect cells using the baculovirus expression system, and complexes were immunoprecipitated using an antibody directed against the p44 subunit of the core-TFIIH in low-salt conditions (buffer B containing 75 mM KCl). After elution with a synthetic peptide recognized by Ab-p44, equal amounts of purified rIIHs were analyzed by SDS/PAGE with 12% (wt/vol) polyacrylamide followed by Western blot (WB) analysis with antibodies directed against XPB, the N-terminus of XPD, p44, CDK7, or p8. Arrowheads indicate the theoretical molecular weight of each XPD mutated form. The asterisk indicates a nonspecific band.

Fig. 2.

DNA repair and helicase activities of rIIHs. (A) In vitro double-incision assay. Increasing amounts of immunopurified rIIH were added to an incision/excision assay using recombinant NER factors, and the reaction was analyzed by electrophoresis followed by autoradiography. “t” corresponds to ∼25 ng of rIIH with p44 as reference. (B) The 5′–3′ helicase activity of XPD variants. Equivalent amounts of each Flag-purified XPD variant (∼100 ng) were added to a 5′-strand extension probe obtained by annealing a 52-nt single-strand DNA to a 5′ ³²P-labeled 25-nt single-stranded DNA in the presence of increasing amounts of p44 (0, 50, or 500 ng). Single- and double-stranded DNA are separated by electrophoresis on a 14% (wt/vol) polyacrylamide gel and analyzed by autoradiography (lanes 3–15). The symbols “−” and “Δ” indicate the native and denatured probes, respectively (lanes 1 and 2). (C) DNA-binding activity. Increasing amounts of XPD variants (Left, lanes 1–9) and CAK-XPD (Right, lanes 10–14) were incubated with the labeled 5′-strand extension probe shown in B, and the resulting nucleoprotein complexes were analyzed by electrophoresis using a 6% (wt/vol) polyacrylamide gel followed by autoradiography with XPD as reference. Densitometric analysis of lanes 3, 5, 7, 9, 12, and 14 are shown as below. “×” corresponds to 100 ng of XPD. BP, bound probe; FB, free probe. (D) 5′–3′ helicase activities of rIIH complexes. Insect cells were infected with a set of baculoviruses overexpressing the subunits of TFIIH including either wild-type or mutated Flag-tagged XPD, and complexes were immunoprecipitated using an antibody directed against the Flag epitope in buffer B. After elution with the Flag synthetic peptide, core-TFIIH/XPD (core-IIH, lanes 1–3) and Holo-TFIIH (rIIH, lanes 4–6) were analyzed by Western blot analysis (WB), and equivalent amounts of complex (∼200 ng) were tested for their 5′–3′ helicase activities (Helicase). The negative control is shown in lane 7. Densitometric analysis suggests a fivefold decrease of Holo-TFIIH (lane 4) 5′–3′ helicase activity compared with that of core-TFIIH (lane 1) in the case of XPD-wt.

Fig. 3.

Transcription and CTD kinase activity of rIIHs. (A) Basal transcription activity. Increasing amounts of purified rIIH with different XPD variants were added to an in vitro reconstituted transcription system lacking TFIIH. Transcripts were analyzed by electrophoresis followed by autoradiography and quantification. The length of the corresponding transcript is indicated on the left. Data from a representative experiment are shown in the histogram. For each mutant, the transcription activity from three independent experiments and normalized to wild-type rIIH is indicated. “//” indicates saturation; “t” corresponds to ∼25 ng of rIIH with p44 as reference; au, arbitrary units. (B) To evaluate the RNA Pol II kinase activity of the TFIIH variants, purified core-TFIIH (200 ng), CAK (100 ng), and XPD (x or 2×) mutants were mixed in an in vitro assay containing all the basal transcription factors and the AdMLP. Arrows indicate hypophosphorylated (IIA) and hyperphosphorylated (IIO) forms of RNA Pol II. “×” corresponds to ∼100 ng of XPD. (C) The transcription activity of rIIHs containing the different XPD variants was assessed in presence of increasing amounts of purified recombinant CAK. “+” corresponds to 50 ng of rIIH-wt, -C259Y, and -R722W and 150 ng of rIIH-ΔARCH. Wild-type rIIH was used as control. The signals were quantified and normalized to wild type assuming activities of 30, 17, and 9% for rIIH-C259Y, -R722W, and -ΔARCH, respectively.

Fig. 4.

The ARCH domain is a recruitment platform for CAK. (A) Pairwise interactions between XPD variants and MAT1 or p44. Sf9 insect cells were coinfected with viruses that allow the expression of Flag-tagged XPD variants and a virus that allows the expression of MAT1 or p44. Proteins were immunopurified with the anti-Flag M2 resin (Left) or were immunoprecipitated with an anti-XPD antibody (Right) in the presence of 250 mM NaCl (buffer C). Input and purified complexes were analyzed by anti-p44, anti-MAT1, or anti-Flag Western blot. The asterisk indicates a nonspecific band. (B) Pairwise interactions between XPD variants and XPG. Sf9 insect cells were coinfected with viruses that allow the expression of Flag-tagged XPD variants and a virus that allows the expression of c-myc–tagged XPG. Immunopurification was performed with anti–c-Myc resin in buffer C. Input and the purified complexes were analyzed by Western blot with anti-Flag antibody. (C) Interactions between XPD fragments and CAK. Sf9 insect cells were coinfected with a virus that allows the expression of the CAK subcomplex with CDK7 C-terminal Strep-tag fusion and with viruses that allow the expression of N-terminal XPD (residues 1–245), C-terminal XPD (residues 443–762), or ARCH (residues 245–443) with an N-terminal Flag tag. The capacity of XPD fragments to bind CAK and to copurify was tested using the Flag-tag (lanes 1–3) or Strep-tag II (lanes 4–6) for the purification of the complexes in buffer C. Purified proteins were analyzed using PAGE with 12% (wt/vol) polyacrylamide followed by Coomassie staining. Proteins are indicated by arrowheads. (D) Consequences of XPD mutations on association with CAK and core-TFIIH. Insect cells were infected with a set of baculoviruses overexpressing the subunits of TFIIH including either wild-type or mutated Flag-tagged XPD, and complexes were immunoprecipitated using an antibody directed toward the Flag epitope in buffer C. After elution with the Flag synthetic peptide, immunopurified complexes (rIIHs) were analyzed by Western blot analysis (WB, Upper) and were tested for their capacity to phosphorylate the CTD of RNA Pol II (autoradio, Lower). Experiments were performed in triplicate. Histograms represent estimated ratios (in arbitrary units, au) between MAT1 (a representative CAK subunit) and XPD or p44 (a representative core-TFIIH subunit).

Fig. 5.

Implications of XPD in retinoic acid-dependent recruitment of TFIIH on the RARβ2 promoter. (A) Relative RARβ2 mRNA expression monitored by qPCR from XPD-transfected HD2 cells (XPD-wt, -C259Y, -R722W) treated with 1 µM t-RA. The effect of XPD mutations on nuclear hormone receptors mediated transactivation. The XPD-deficient cell line HD2 was transfected with an empty plasmid (blue diamond) or with a plasmid expressing XPD-wt (red square), XPD-C259Y (green triangle), or XPD-R722W (magenta cross) and was treated with t-RA. Transcription of the RARβ2 mRNA was quantified by qPCR. (B) HEK293 Flp-IN cells were stably transfected with 3Flag-XPD (XPD-wt, -C259Y, -R722W). Chromatin extracts were prepared and analyzed for Flag, XPD, and MAT1 by Western blot (Left). Whole-cell extracts were immunoprecipitated using the Flag tag and analyzed for XPD, p44, and MAT1 (Right). (C–H) ChIP/ReChIP monitoring the coimmunoprecipitation of the RARβ2 promoter using Flag or XPD antibodies (C–E) or a combination of antibodies against Flag/MAT1, Flag/p44, or Flag/pol II (F–H ) from 3Flag-XPD–transfected HEK293 stable cell lines (XPD-wt, XPD-C259Y, XPD-R722W) treated with t-RA (1 µM).

Fig. 6.

XPD structure and TFIIH architecture. (A) Modular organization of XPD. Thermoplasma acidophilum XPD (Protein Data Bank ID code 2VSF) is composed of four structural domains: two RecA-like helicase domains (HD1 in blue and HD2 in magenta), one domain that contains an iron/sulfur group (in cyan), and a domain described as the ARCH domain (in green). The C-terminal extension of eukaryotic XPD that is required for interaction with the p44 core-TFIIH subunit and for which no structural data are available is represented by an ellipse. Residue 259 mutated into a tyrosine in the TTD12PV patient cell line maps onto the first helix of the ARCH domain, and its side chain points into the center of the helical bundle. (B) Schematic of subunit architecture of TFIIH. Each subunit is represented by a circle with the radius of a sphere corresponding to its molecular weight. Interactions between XPB, p52, and p8/TTD-A stimulate XPB ATPase activity and consequently favor binding of TFIIH to damaged DNA. Interactions between p44 and XPD stimulate the helicase activity, allowing unwinding of DNA around the lesion and subsequent double incision by the endonucleases XPF-ERCC1 and XPG. XPD helicase activity, dispensable for transcription initiation but required for NER, is repressed when CAK is associated with TFIIH.

Fig. P1.

The XPD helicase, a subunit of transcription/DNA repair factor TFIIH, catalyzes the unwinding of damaged DNA during NER and bridges core-TFIIH to the CAK kinase complex. We have established that the ARCH domain of XPD constitutes a platform for the recruitment of CAK through its menage a trois 1 (MAT1) subunit and analyzed the C259Y mutation identified in patients with TTD. This mutation not only affects the interaction with MAT1, thereby decreasing the in vitro transcription/phosphorylation activities of TFIIH itself and impeding the efficient recruitment of the transcription machinery on the promoter of the RARβ2 gene, but also impairs the helicase activity of XPD and the NER activity of TFIIH. Together, these results identify the ARCH domain of XPD as a platform for the recruitment of CAK and show that it plays a key role in DNA recognition and in the regulation of the helicase activity.