A disease-associated XPA allele interferes with TFIIH binding and primarily affects transcription-coupled nucleotide excision repair

Abstract

XPA is a central scaffold protein that coordinates the assembly of repair complexes in the global genome (GG-NER) and transcription-coupled nucleotide excision repair (TC-NER) subpathways. Inactivating mutations in XPA cause xeroderma pigmentosum (XP), which is characterized by extreme UV sensitivity and a highly elevated skin cancer risk. Here, we describe two Dutch siblings in their late forties carrying a homozygous H244R substitution in the C-terminus of XPA. They present with mild cutaneous manifestations of XP without skin cancer but suffer from marked neurological features, including cerebellar ataxia. We show that the mutant XPA protein has a severely weakened interaction with the transcription factor IIH (TFIIH) complex leading to an impaired association of the mutant XPA and the downstream endonuclease ERCC1-XPF with NER complexes. Despite these defects, the patient-derived fibroblasts and reconstituted knockout cells carrying the XPA-H244R substitution show intermediate UV sensitivity and considerable levels of residual GG-NER (~50%), in line with the intrinsic properties and activities of the purified protein. By contrast, XPA-H244R cells are exquisitely sensitive to transcription-blocking DNA damage, show no detectable recovery of transcription after UV irradiation, and display a severe deficiency in TC-NER-associated unscheduled DNA synthesis. Our characterization of a new case of XPA deficiency that interferes with TFIIH binding and primarily affects the transcription-coupled subpathway of nucleotide excision repair, provides an explanation of the dominant neurological features in these patients, and reveals a specific role for the C-terminus of XPA in TC-NER.

Keywords: DNA repair; nucleotide excision repair; transcription factor II H; transcription-coupled repair; xeroderma pigmentosum protein A.

Fig. 1.

Patient or rescue cells expressing XPA^H244R show residual UDS. (A) Sequence confirmation of the H244R amino acid substitution in genomic DNA of patient XP2LD compared to WT DNA (48BR). (B) Schematic representation of the XPA protein with its interaction domains. (C) XPA protein expression in primary fibroblasts from 48BR (WT), XP2LD (XPA^H244R), and XP1PD (XPA^C108F) by western blot. RNAPII-S2 and TFIIH/p89 levels are used as loading controls. (D) Expression levels of endogenous XPA in U2OS(FRT) WT and U2OS(FRT) XPA-KO cells, and GFP-XPA expression in U2OS(FRT) XPA-KO cells reconstituted with GFP-XPA^WT, GFP-XPA^H244R, or GFP-XPA^C108F. CSB, CSA, TFIIH/p89, and RNAPII-S2 are used as loading controls. (E) Sequence confirmation of the H244R or C108F amino acid substitution in the U2OS(FRT) XPA-KO cells reconstituted with GFP-XPA^WT, GFP-XPA^H244R, or GFP-XPA^C108F cDNA. (F) Clonogenic survival assay after irradiation with various doses of UV-C in U2OS(FRT) WT, XPA-KO, and GFP-XPA reconstituted cell lines. The bars are the average of two experiments. The error bars reflect the SD. (G) Representative images and (H) quantification of EdU incorporation in the indicated primary fibroblasts after 30 J/m² UV-C irradiation through 5-µm pore membranes. Sites of local damage are identified by CPD staining. The EdU signal has been normalized for CPD levels at sites of local damage. (I) Representative images and (J) quantification of EdU incorporation in the indicated RPE1 cells deficient in TC-NER (CSB-KO cells) to specifically measure UDS by GG-NER after 30 J/m² UV-C irradiation through 5-µm pore membranes. Sites of local damage are identified by CPD staining. The EdU signal has been normalized for CPD levels at sites of local damage. (H and J) All cells are depicted as individual data points with the bar representing the mean of all data points. The individual means of four biological replicates are depicted as gray points with black circles.

Fig. 2.

The H244R mutation in XPA reduces in vitro NER activity. (A) Purification of XPA^H244R protein. Two fractions from purification, #7 and #10 were tested for in vitro assays. (B) In vitro NER activity of XPA^WT and XPA^H244R mutant using purified NER proteins. A plasmid containing a site-specific AAF lesion was incubated with purified XPA (20 nM), XPA-RAD23B (5 nM), TFIIH (10 nM), RPA (42 nM), XPG (27 nM), and XPF-ERCC1 (13 nM) for 0 to 90 min. The excision products were detected by annealing to a complementary oligonucleotide with a 4dG overhang, which was labeled with [α-32P] dCTP. (C) Quantification of (B). Two independent experiments were performed.

Fig. 3.

XPA^H244R has a weakened association with TFIIH after UV. (A) Volcano plot depicting the UV-specific enrichment of proteins after pull-down of GFP-XPA^WT at 1 h after irradiation with 20 J/m² UV-C versus (unirradiated) GFP-NLS analyzed by label-free MS. The enrichment (log₂) is plotted on the x-axis and the significance (2-sided t test -log₁₀ P-value) is plotted on the y-axis. Highlighted are XPA (purple) and NER proteins (blue). (B) As in a, but for pull-down of GFP-XPA^H244R at 1 h after irradiation with 20 J/m² UV-C compared to unirradiated, mock-treated GFP-NLS. (C) Coimmunoprecipitation of GFP-XPA or GFP-NLS from U2OS(FRT) WT cells expressing GFP-NLS or U2OS(FRT) XPA-KO cells reconstituted with GFP-XPA^WT or GFP-XPA^H244R, either without or after UV irradiation (20 J/m² UV-C). The input is 0.75% of the total protein lysate.

Fig. 4.

Reduced accumulation of XPA^H244R impairs ERCC1-XPF recruitment at UV lesions. (A) Representative images of the recruitment of indicated GFP-tagged XPA variants to UV-C laser tracks, measured by live cell imaging. (B) Quantification of (A). Data represents average and SD of three independent experiments of 25 to 78 cells per experiment. (C) Representative images of the recruitment of indicated GFP-tagged XPA mutants reconstituted in U2OS(FRT) XPA-KO cells, to sites of damage after 30 J/m² UV-C irradiation through 5-µm pore membranes. Sites of local damage are identified by TFIIH/p89 staining. (D) Quantification of (C). Quantification of the recruitment of TFIIH/p89 is shown in SI Appendix, Fig. S5B.  (E) Representative images and (F) quantification of ERCC1 recruitment in the indicated primary fibroblasts after 30 J/m² UV-C irradiation through 5-µm pore membranes. Sites of local damage are identified by TFIIH/p89 staining. Quantification of the recruitment of TFIIH/p89 is shown in SI Appendix, Fig. S5C.  (G) Representative images and (H) quantification of ERCC1 recruitment in the indicated U2OS(FRT) cells after 30 J/m² UV-C irradiation through 5-µm pore membranes. Sites of local damage are identified by TFIIH/p89 staining. Quantification of the recruitment of TFIIH/p89 is shown in SI Appendix, Fig. S5D. (D, F, and H) All cells are depicted as individual data points with the bar representing the mean of all data points. The individual means of three biological replicates are depicted as colored points with black circles.

Fig. 5.

Patient or rescue cells expressing XPA^H244R are defective in TC-NER. (A) Clonogenic survival assay after 72 h treatment with various doses of Illudin S in U2OS(FRT) WT, XPA-KO, and GFP-XPA reconstituted cell lines. The bars are the average of three to four experiments. The error bars reflect the SD. (B) Clonogenic survival assay after continuous treatment with various doses of Trabectedin in U2OS(FRT) WT, XPA-KO, and GFP-XPA reconstituted cell lines. The bars are the average of two to three experiments. The error bars reflect the SD. (C) Representative images of indicated primary fibroblasts pulse-labeled with 5-ethynyl-uridine (5-EU). Cells were either mock-treated, irradiated with 9 J/m² UV-C, or exposed to 30 ng/mL Illudin S for 3 h. Cells were subsequently pulse-labelled with 5-EU pulse-labeled at the indicated timepoints. (D) Quantification of RNA synthesis from (C), relative to the mock condition per cell line. (E) Representative images and (F) quantification of EdU incorporation in the indicated RPE1 cells deficient in GG-NER (XPC-KO cells) to specifically measure UDS by TC-NER after 100 J/m² UV-C irradiation through 5-µm pore membranes. Sites of local damage are identified by CPD staining. (D and F) All cells are depicted as individual data points with the bar representing the mean of all data points. The individual means of three to four biological replicates are depicted as points with black circles.