Fig 2: XPC and TFIIH are continuously recruited to UV damage in XPCS patient fibroblasts. (A) Representative immunofluorescence pictures showing the co-localization of XPF and XPB at LUD in patient fibroblasts XPCS1CD and CS1USAU, compared to wild type C5RO fibroblasts. Cells were stained 1 h and 8 h after local UV irradiation (60 J/m2) through an 8 μm microporous filter with antibodies against XPF and XPB. (B) Representative immunofluorescence pictures showing the LUD recruitment of XPD and XPC in fibroblasts derived from patients carrying mutations in XPB, XPD, XPG, XPF and XPA, affected by mild XP (XP42RO, XP32BR, XP6BE), XPCS (XPCS1CD, XPCS1BA, XPCS2, XPCS1RO) or DSC (XP25RO) compared to wild type C5RO fibroblasts. Cells were locally UV irradiated with 60 J/m2 UVC through an 8 μm microporous filter and stained 1 h and 8 h after irradiation with antibodies against XPD and XPC. Scale bar: 5 μm.

Fig 3: New H2A.X Accumulation at UV Sites and Replication Foci Is Coupled to DNA Synthesis(A) Scheme: XPG’s role in the late steps of UVC damage repair. Percentage of cells showing repair synthesis (EdU) at UVC-damage sites (CPDs [cyclobutane pyrimidine dimers]) in U2OS cells treated with the indicated siRNAs (siLUC, control). At least 200 cells were scored in 2 independent experiments.(B and C) New H2A.X accumulation at UVC damage sites marked by XPA or XPB analyzed 2 hr after irradiation in the presence of flavopiridol (+FLV) in U2OS H2A.X-SNAP cells treated with siRNAs targeting XPG (B) or PCNA (C) (siLUC, control). Knockdown efficiencies are verified by western blot and by impaired CAF-1 recruitment to damage sites.(D) New H2A.X accumulation at replication foci (marked by EdU before aphidicolin addition) in U2OS H2A.X-SNAP cells treated with flavopiridol (+FLV) and the replication inhibitor aphidicolin (Aphi). Replication inhibition is shown by impaired CAF-1 recruitment to EdU foci.Bar charts show mean ± SD from at least 2 independent experiments. Scale bars, 10 µm. ns, non-significant; *p < 0.05; **p < 0.01; ***p < 0.001. See also Figure S4.

Fig 4: Summary of the mechanism of trabectedin-induced TC-NER-mediated SSB formation and toxicity, and the development of TRABI-Seq.Trabectedin-DNA adducts are exclusively recognized by TC-NER, and not by GG-NER. The adducts then block the incision of the XPG endonuclease, causing persistent XPF-mediated breaks. Mapping those breaks in a genome-wide fashion displays where TC-NER is active: predominantly in highly transcribed genes and their upstream regions due to a link to divergent transcription.

Fig 5: Trabectedin-induced DNA break formation and toxicity depend on the catalytic activity of XPF but not that of XPG.a HAP1 WT, ERCC1-KO, and XPF-D687A cells were treated with trabectedin for 2 h and colonies counted after 7 days. Mean ± SEM of 5 (WT, XPF-D687A) and 3 (ERCC1-KO) biological replicates (3 technical replicates per experiment). Provided p-values are derived using ordinary two-way ANOVA with Dunnett’s multiple comparisons test (between WT and ERCC1-KO or XPF-D687A at each concentration). b HAP1 WT, ERCC1-KO, and XPF-D687A cells were arrested in G1 with palbociclib and treated with trabectedin (50 nM, 2 h) and incubated for up to 4 h with or without repair synthesis inhibitors (0.5 mM HU, 5 μM AraC). ssDNA breaks were analyzed by alkaline COMET chip assays. Mean ± SEM of 3 biological replicates (Supplementary Fig. 2c). Provided P-values are derived using two-tailed paired t-test (between WT and ERCC1-KO or XPF-D687A at each recovery time). c XP2YO patient cells were treated with trabectedin for 2 h and colonies were counted after 8 days. Mean ± SEM of 2 biological replicates (3 technical replicates per experiment). d U2OS WT and XPF-KO cells were arrested in G1 with palbociclib and treated with trabectedin (50 nM, 2 h) and allowed to recover for up to 4 h with or without repair synthesis inhibitors (1 mM HU, 10 μM AraC). ssDNA breaks were analyzed by alkaline COMET chip assays. Mean ± SEM of 4 biological replicates. P values were derived using ordinary two-way ANOVA with uncorrected Fisher’s LSD (between WT and XPF-KO at each recovery time). e HAP1 WT, XPG-KO, and XPG-E791A cells were treated with trabectedin for 2 h and colonies counted after 7 days. Mean ± SEM of 4 biological replicates (3 technical replicates per experiment). Provided P-values are derived using ordinary two-way ANOVA with Dunnett’s multiple comparisons test (between WT and XPG-KO or XPG-E791A at each concentration). f HAP1 WT, XPG-KO, and XPG-E791A cells were arrested in G1 with palbociclib (2 µM, 24 h) and treated with trabectedin (50 nM, 2 h). Cells were kept in G1 with or without repair synthesis inhibitors (0.5 mM HU, 5 μM AraC) and incubated for up to 4 h. ssDNA breaks were analyzed by alkaline COMET chip assays. Mean ± SEM of 5 biological replicates (Supplementary Fig. 3d–e). Provided P-values are derived using ordinary two-way ANOVA with Tukey’s multiple comparisons test (between WT and XPG-KO or XPG-E791A at each recovery time). g A simplified schematic of assessing NER incision activity on trabectedin-induced DNA adducts in HAP1 WT, XPF-D687A, ERCC1-KO, XPG-E791A, and XPG-KO cells using alkaline COMET chip assays. Source data are provided as a Source Data file.