In TFIIH, XPD helicase is exclusively devoted to DNA repair

Abstract

The eukaryotic XPD helicase is an essential subunit of TFIIH involved in both transcription and nucleotide excision repair (NER). Mutations in human XPD are associated with several inherited diseases such as xeroderma pigmentosum, Cockayne syndrome, and trichothiodystrophy. We performed a comparative analysis of XPD from Homo sapiens and Chaetomium thermophilum (a closely related thermostable fungal orthologue) to decipher the different molecular prerequisites necessary for either transcription or DNA repair. In vitro and in vivo assays demonstrate that mutations in the 4Fe4S cluster domain of XPD abrogate the NER function of TFIIH and do not affect its transcriptional activity. We show that the p44-dependent activation of XPD is promoted by the stimulation of its ATPase activity. Furthermore, we clearly demonstrate that XPD requires DNA binding, ATPase, and helicase activity to function in NER. In contrast, these enzymatic properties are dispensable for transcription initiation. XPD helicase is thus exclusively devoted to NER and merely acts as a structural scaffold to maintain TFIIH integrity during transcription.

Figure 1. XPD and its partners in TFIIH.

Schematic representation of XPD and its interaction partners p44 and MAT1 within the CAK complex of TFIIH. XPD from taXPD in the center is shown with the two helicase domains (HD1 and HD2) in yellow and red, respectively, with the FeS cluster-containing domain in cyan and the Arch domain in green. P44, which interacts with the C-terminal end of XPD, is shown in pale green The CAK complex, which interacts with XPD via the MAT1 subunit, is shown in different blue colors.

Figure 2. Functional analysis of ctXPD.

(a) SEC of ctXPD wild type with ctp44 (1–285). Ctp44 (pink) and ctXPD (blue) were analyzed separately and in a 1∶1 stoichiometry (red) mixed prior to SEC. (b) SEC of the ctK719W variant with ctp44 (1–285). The color coding is chosen as in (a). (c) ssDNA binding of ctXPD was analyzed by biolayer interferometry. Measurements were performed in triplicate and with different protein batches. (d) In vitro ATPase assay of ctXPD wild type in the presence (black bars) and absence of ctp44 (1–285) (grey bar). Subsequently all variants were analyzed in the presence of ctp44 (1–285). The data used to generate panels (c) and (d) have been deposited as supplementary information in xls format (Table S1).

Figure 3. Helicase activity of ctXPD and hsXPD and its variants.

(a) Helicase activity of wild-type ctXPD in the presence of ctp44 (1–285) and its variants analyzed utilizing a fluorescence-based helicase assay. (b) hsXPD wild-type and variants were expressed in insect cells using the baculovirus overexpression system and immunoprecipitated using an antibody (Ab) directed toward the Flag epitope fused at the N terminus of the protein. After elution with the Flag synthetic peptide, equal amounts of purified proteins were resolved by SDS/PAGE with 12% (w/v) polyacrylamide followed by Western blot (WB) analysis (upper panel, lanes 3 to 12). Bands shown in lanes 2 and 13 in the upper panel are a duplication of lanes 3 (wild type) and 11 (L372A). Purified XPD wild type and variants were added to a 5′-strand extension probe in the presence of an excess of p44 to evaluate their 5′–3′ helicase activity. The reaction was analyzed by electrophoresis on a 14% (w/v) polyacrylamide gel and analyzed by autoradiography. Controls include reactions performed in the absence of wild-type XPD (lane 1) and in the absence of p44 (lane 2). The native and denatured probes have also been analyzed (lanes 14 and 15, lower panel). The data used to generate panels (a) and (b) have been deposited as supplementary information in xls format (Table S2).

Figure 4. NER activity of hsXPD and its variants.

(a) Purified core-TFIIH (rIIH6) resolved by SDS-PAGE followed by Coomassie staining and Western blot analysis. (b and c) NER activity of reconstituted TFIIH containing mutant XPD proteins. XPD wild type or variants (100 and 200 ng) were mixed with purified core-TFIIH (rIIH6) and added to an in vitro double-incision assay using recombinant NER factors. The reaction was analyzed by electrophoresis followed by autoradiography. Incision activities from three independent experiments were quantified and normalized to wild type. (d and e) Host cell reactivation activity of XPD variants. HD2 fibroblasts were transfected with a reporter plasmid expressing firefly luciferase previously exposed to 1,000 J/cm² UVC-light (254 nm) (d) or with the nonirradiated control (e) in combination with vector expressing renilla luciferase to normalize transfection efficiencies and pIERS2-EGFP expressing XPD wild-type or mutant proteins. The firefly luciferase activity in cell lysates (48 h posttransfection), normalized with the internal renilla luciferase standard, assesses repair complementation. The values of three independent experiments are presented as percentages, with 100% being the level of luciferase activity obtained with wild-type XPD. The data used to generate panels (c), (d), and (e) have been deposited as supplementary information in xls format (Table S3).

Figure 5. Transcriptional activity of hsXPD and its variants.

(a) Basal transcription activity. Increasing amounts of the recombinant XPD variants (∼50, 100, and 250 ng) were mixed with purified core-TFIIH (rIIH6) (250 ng) and CAK (300 ng) to an in vitro reconstituted transcription system containing all the basal transcription factors and the AdMLP. Transcripts were analyzed by electrophoresis followed by autoradiography. The length of the corresponding transcript is indicated on the right side. (b) Dose–response curves for XPD-wt and mutants C134S, F193A, R196A/E, and R722W. We have selected the hsF193A and hsR196A/E variants that exhibited levels of transcription similar to wild-type hsXPD as a reference. The transcription activity was estimated from densitometric analysis of autoradiograms (arbitrary units). The data are also plotted as % wild-type activity in the panel to the right. (c) CTD phosphorylation of RNAP II by reconstituted TFIIH with XPD variants was analyzed by SDS-PAGE and followed by WB detection. Purified core-TFIIH (250 ng), CAK (300 ng), and XPD variants (100 or 300 ng) 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 RNAP II. (d) Transcription activity of reconstituted TFIIH containing XPD variants (250 ng core-TFIIH and 200 ng XPD) was assessed in the presence of different amounts of purified recombinant CAK (150 and 300 ng).

Figure 6. Model of the different XPD and XPB functionalities in NER and transcription.

(a) In NER, XPD and XPB act as enzymes and have to mediate protein–protein interactions. XPD ,, and XPB ,, are shown as a cartoon with their molecular surface. The other subunits are depicted schematically. (b) As in (a), with the exception that the cartoon for XPD has been omitted and XPD is shown in green to exemplify that only the “shell” of XPD is needed for transcription.