Fig 2: Oncogenic stress-induced R-loop accumulation drives the FANCA-PARPi synthetic lethality.a, IF staining of R-loops using the S9.6 antibody was performed in the indicated hTERT RPE-1 cells. R-loop intensity per cell was quantified by ImageJ. Data represent mean ± SEM (n=3 biologically independent experiments) and were analyzed by unpaired two-tailed t-test. **** p<0.0001. b, IF analysis of gH2AX foci in the indicated hTERT RPE-1 cells. Cells were transfected with an RNase H1 construct for one day, followed by treatment with Olaparib (10 μM) for 4 hours or no treatment. Doxycycline (200 ng/ml) was used to induce c-Myc expression. Data represent mean ± SEM, analyzed by an unpaired, two-tailed t-test. **p < 0.01, ***p < 0.001. c, IF staining of R-loops was conducted in the indicated MDA-MB-231 cells. Cells were transfected with an RNaseH1 construct for one day, followed by treatment with Olaparib (10 μM) for 4 hours or no treatment. R-loop intensity was quantified by ImageJ. Data represent mean ± SEM (n=2 biologically independent experiments), assessed by an unpaired, two-tailed t-test. * p<0.05, ***p<0.001, **** p<0.0001. d, Schematic representation of the experimental procedure for DRB treatment. e, The indicated MDA-MB-231 cells were synchronized with 2 mM thymidine for 18 hours and released into fresh medium containing either Olaparib (10 μM) and/or DRB (75 μM) for 3.5 hours, as indicated. Representative IF images of gH2AX foci are shown (left). Quantification of the gH2AX foci number is plotted (right). Data represent mean ± SEM (n=3 biologically independent experiments) and were analyzed by unpaired, two-tailed t-test. ***p<0.001, **** p<0.0001. Scale bars, 10 μm. f, Proposed model illustrating the role of FANCA in FEN1-mediated RNA flap cleavage during Okazaki fragment maturation in the presence of oncogene-induced post-replication R-loops on the lagging strand. DRB: 5,6-Dichloro-1-β-D-ribofuranosylbenzimidazole.

Fig 3: The FANCA C-terminal domain is required for FEN1 recruitment and Okazaki fragment maturation.a, b, FANCA deficiency impairs nascent DNA strand integrity. a, Schematic of the BrdU comet assay protocol. MDA-MB-231 cells with the indicated genotypes were either untreated or treated with Olaparib (10 μM). After BrdU pulse-labeling and chase, nascent single strands were assessed using anti-BrdU antibody staining in alkaline comet assays. b, Representative images (left) and quantification of BrdU+ comet tail moments (right) are shown. Data are presented as median ± interquartile range (n=3 biologically independent experiments), analyzed by unpaired, two-tailed t-test. **p<0.01, ****p<0.0001. Scale bars, 50 μm. c, FANCA deficiency increases PARP1 chromatin association. MDA-MB-231 cells with the indicated genotypes were untreated or treated with Olaparib (10 μM) for 4 hours, followed by nuclear fractionation. Western blot analysis (left) quantified PARP1 levels in chromatin, normalized to histone H3 (right). Data are mean ± SEM (n=3 independent biological replicates), analyzed by unpaired, two-tailed t-test. *p<0.05, **p<0.001. d, FANCA deficiency increases ADP-ribosylation. ADP-ribose was detected by IF staining using ADP-ribose (MAR/PAR) monoclonal antibody in MDA-MB-231 cells with the indicated genotypes with 2.5 mM hydroxyurea (HU) treatment for 20 hours. The IR-treated sample served as a positive control. Data shown are mean ± SEM, and statistical significance was assessed by unpaired two-tailed t-test. **p<0.01. e,f, The C-terminal domains (D4 and D5) of FANCA mediate FEN1 interaction. e, Schematic illustration of full-length (FL) and deletion mutants of FANCA used in this study. FANCA constructs were cloned into inducible PiggyBac expression vectors. f, Co-IP experiment with anti-Flag antibody to pull down Flag-tagged proteins using whole cell lysate and Western blot were performed as indicated. g, FANCA-FANCG-FAAP20 subcomplex cryoEM structure model (PDB #7KZP) with color-coded D1 – D5 subdomains. h,i Functional relevance of FANCA domains. Clonogenic survival assays were conducted on FANCA−/− MDA-MB-231 cells reconstituted with FANCA FL or DD1-D5 mutants treated with Olaparib (h) or mitomycin C (i). Data are mean ± SEM (n=3 biologically independent replicates). j, FANCA DD4 and DD5 mutants impair PARPi-induced DNA damage repair. IF analysis of gH2AX foci in FANCA−/− MDA-MB-231 cells reconstituted with FL or DD1-D5 mutants, either untreated or treated with Olaparib (10 μM) for 4 hours, followed by 24-hour recovery. Data are mean ± SEM, analyzed by unpaired, two-tailed t-test. **p<0.01, ***p<0.001, **** p<0.0001. k,l FANCA promotes FEN1 recruitment. (k) Representative IF images of FEN1 and PCNA in MDA-MB-231 cells with the indicated genotypes, untreated or treated with Olaparib (10 μM) for 4 hours. Quantification of FEN1 foci (right) is shown. Data are mean ± SEM (n=4 biologically independent experiments), analyzed by unpaired two-tailed t-test. ***p<0.001, **** p<0.0001. Scale bars, 10 μm. l, IF analysis of FEN1 foci in FANCA−/− MDA-MB-231 cells reconstituted with FL, ΔD4, or ΔD5 mutants under the same conditions. Data are mean ± SEM (n=3 biologically independent experiments), analyzed by unpaired two-tailed t-test, ***p<0.001, **** p<0.0001. ΔD1- ΔD5: deleted domains 1–5.

Fig 4: Lentiviral exposure enables the detection of lagging strand ssDNA gaps by the S1 DNA spreading assay independently of the vector backbones and transgenes.U2OS cells were infected with lentiviral particles encoding either shLUC in the pLKO.1 vector (A) or YFP in the pFLRu-FH vector (B). Four days after infection, cells were subjected to the S1 DNA spreading assay with and without FEN1 inhibitor and analyzed as in Fig.1. (C) U2OS cells were exposed to conditioned media lacking or containing shLUC lentivirus five days prior to the S1 DNA fiber spreading assay with FEN1 inhibitor. (D) U2OS cells were infected or not with 0.5 μl of purified shLUC lentivirus (MOI of 5) diluted by 1 ml fresh medium and subjected to the S1 DNA fiber spreading assay with FEN1 inhibitor seven days later. More than 150 dual-colored fibers were quantified for each biological condition. Three independent experiments were performed showing similar trends. P values were based on Kruskal–Wallis test with Dunnett’s multiple comparisons.

Fig 5: Single-stranded DNA gaps due to FEN1 loss-of-function by lentiviral shRNA infection but not chemical inhibition can be detected by DNA fiber spreading assay.(A) U2OS cells were sequentially labeled with 30 μM CldU for 25 min and 250 μM IdU for 45 min during which time 10 μM FEN1 inhibitor LNT1 was present or absent. Cells were subsequently subjected to the S1 DNA fiber spreading analysis. (B) U2OS cells were infected with lentiviral particles encoding shRNA sequences targeting firefly luciferase (shLUC) or human FEN1 (shFEN1), and subjected to the S1 DNA fiber spreading assay after four days. Western blots for FEN1 and tubulin are shown. More than 150 dual-colored fibers were quantified for each biological condition. Three independent experiments were performed which showed similar trends. P values were based on Kruskal–Wallis test with Dunnett’s multiple comparisons.