Fig 2: DDR at ALT telomeres induced by FANCM deficiency is mediated by XPF.a Representative images of TRF2 and XPF co-immunostaining in U2OS cells transfected with FANCM siRNAs for 3 days. Arrows indicate the colocalization events. b Quantification of colocalization events in (a). The number of XPF-TRF2 colocalization per nucleus (left). The intensity of XPF foci that are colocalized with TRF2 (right). P-values by two-sided Mann-Whitney test. Bars, mean ± SEM. n, cell number (left) or number of XPF foci (right). Data of three independent experiments. c DRIP-qPCR to detect telomeric R-loops in U2OS cells after transfection with control, XPF, or FANCM siRNAs for 3 days. Relative R-loop levels were normalized to the same amount of genomic DNA treated with RNase H prior to DRIP. P-values by two tailed Student’s t-test. Bars, mean ± SD. Representative of three independent experiments. Other replicates show similar trends and are provided in the Source Data file. d Quantification of APBs in U2OS cells transfected with siRNAs. APB foci were determined by large TRF2 foci with PML staining shown in Supplementary Fig. 5d. n, cell number. Bars, mean ± SEM. P-values by two-sided Mann-Whitney test. Data of three independent experiments. e Representative images of immuno-DNA FISH to detect the co-localization of ?H2AX and telomeres in U2OS cells after transfection with control, XPF, or FANCM siRNAs for 3 days. Arrows indicate the colocalization events. f Quantification of ?H2AX foci at telomeres in (e). n, cell number. Bars, mean ± SEM. P-values by two-sided Mann-Whitney test. Data of two independent experiments. g Quantification of top 5% telomere intensity foci in (e). n, cell number. Bars, medians. P-values by two-sided Mann-Whitney test. Representative of three independent experiments. Other replicates show similar trends and are provided in the Source Data file. a–g Mean or median values of each group shown on the top of figures.

Fig 3: XPF is recruited by TERRA R-loops to generate DNA damage response.a Representative images of XPF and TRF2 staining in HeLa, WI38-VA, and U2OS cells. U2OS cells were expressing wildtype RNase H1 or catalytic-dead mutant RNase H1. b Quantification of the percentage of TRF2 colocalized with XPF per nucleus in various cell lines and in U2OS cells expressing WT and mutant RNase H1. Percentage of TRF2 co-localized with XPF = (XPF-TRF2 co-localization events / total TRF2 foci). Data of three independent experiments. c Quantification of the percentage of TRF2 colocalized with XPF per nucleus in U2OS cells expressing RCas9-sgRNAs. Data of three independent experiments. Other replicates show similar trends and are provided in the Source Data file. d Representative images of immuno-DNA FISH to detect the colocalization of ?H2AX and telomeres in U2OS cells after 72 h transfection with control or XPF siRNAs. Arrowheads indicate colocalization events. e Western blot analysis for XPF in U2OS cells after siRNA knockdown. GAPDH, loading control. f Quantification of ?H2AX foci at telomeres. Data of three independent experiments. g Representative images of immuno-DNA FISH to detect the co-localization of RPA70 and telomeres in U2OS cells after transfection with control or XPF siRNAs. Arrowheads indicate colocalization events. Data of three independent experiments. h Quantification of RPA70 foci at telomeres. Data of three independent experiments. a–h Mean or median values of each group shown on the top of figures. P-values by two-sided Mann-Whitney test. Bars, mean ± SEM. n, cell number.

Fig 4: XPF promotes HR and break-induced telomere synthesis.a Representative images of TRF2 and BRCA1 immunostaining in U2OS cells transfected with control, XPF, FANCM, or XPF/FANCM siRNAs for 3 days. b Quantification of colocalization events of BRCA1 and TRF2 in (a). c Quantification of colocalization events of RAD51 and TRF2 in U2OS cells transfected with control, XPF, FANCM, or XPF/FANCM siRNAs for 3 days. d Representative images of EdU-DNA-FISH to detect telomere synthesis in siRNA transfected U2OS cells after the treatment of RO-3306. Arrows indicate the colocalization events. e Quantification of EdU and telomere colocalization in non-S phase cells in (d). f Quantification of POLD3 and TRF2 colocalization events in U2OS cells transfected with siRNAs after the treatment of RO-3306. g Quantification of EdU and telomere colocalization events in stable cell lines expressing RCas9-sgRNAs. TERRA knockdown cells (sgTER_1C6, sgTER_1C21) or control cells (sgSense, sg?2). h Quantification of EdU and telomere colocalization events in U2OS cells with transient TERRA knockdown by viral transduction with RCas9-sgRNA plasmids. i Quantification of EdU and telomere colocalization events in WI38-VA cells transfected with control, XPF, FANCM, or XPF/FANCM siRNAs. a–i Data of three independent experiments. n, cell number. Bars, mean ± SEM. Mean values of each group shown on the top of figures. P values by two-sided Mann-Whitney test.

Fig 5: Decreased activity of the nucleotide excision repair (NER) pathway in cells with prolonged p21WAF1/Cip1 expression. a RNAseq analysis showed that essential factors of the NER pathway were statistically significantly down-regulated (p = 0.05; see also Additional file 1: Figure S3 for specific real-time RT-PCR validation) in 96-h induced Saos2- (i) and Li-Fraumeni- (ii) p21WAF1/Cip1 Tet-ON cells. Selective immunoblots for DDB1, ERCC1, ERCC4-XPF, XPC, and ERCC5-XPG, key factors in NER [22], confirming the specificity of the RT-PCR results. a-Tubulin served as loading control; the same protein extracts were used as in Fig. 2. b Decreased repair capacity of N-alkylpurine monoadducts in induced Saos2- (i) and Li-Fraumeni- (ii) p21WAF1/Cip1 Tet-ON cells and treated with monohydroxymelphalan, an inducer of specific NER substrates [69–71]. The data shown are based on five independent experiments with at least two analyses for independent experiment/independent experiment experiment (* p < 0.05, error bars indicate SDs). The middle panel depicts the components and function of NER. The NER pathway is responsible for repair of bulky lesions, especially UV-induced thymine dimers and 6,4-photoproducts, as well as non-bulky ones. Following DNA damage recognition, a short single-stranded DNA fragment that contains the lesion is removed. The remaining undamaged single-stranded DNA segment is used by DNA polymerase as a template to synthesize the complementary sequence. Final ligation to complete NER and formation of a double-stranded DNA is carried out by DNA ligase. Depending on how the DNA damage is recognized, NER can be divided into two subpathways: transcription coupled NER (TC-NER) and global genome NER (GG-NER). While the two subpathways differ in how they recognize DNA damage, they share the same process for lesion incision, repair, and ligation. RBX1 Ring-box 1, Cul4 Cullin 4, DDB1/2 Damage specific DNA binding protein 1/2, ERCC8 (CSA) ERCC excision repair 8, CSA ubiquitin ligase complex subunit, ERCC6 (CSB) ERCC excision repair 6, chromatin remodeling factor, USP7 Ubiquitin-specific peptidase 7; ERCC4-XPF Excision repair 4, endonuclease, ERCC5-XPG ERCC excision repair 5, endonuclease, XPA XPA, DNA damage recognition and repair factor, XAB2 XPA binding protein 2, RPA Replication protein A, HMGN1 High mobility group nucleosome binding domain 1; XPC XPC complex subunit, DNA damage recognition and repair factor, RAD23B RAD23 homolog B, CETN2 Centrin 2, CDK7 Cyclin-dependent kinase 7, MNAT1 CDK activating kinase assembly factor, CCNH Cyclin H, TFIIH1–4 Transcription/repair factor IIH 1–4, ERCC3 ERCC excision repair 3, TFIIH core complex helicase subunit, ERCC2 ERCC excision repair 2, TFIIH core complex helicase subunit, TTDA (GTF2H5/TFB5) General transcription factor IIH subunit 5