Fig 2: PCNA unloading defects are responsible for fork degradation in BRCA-deficient cells.a, b SIRF assay showing deficient PCNA unloading from nascent DNA upon replication fork arrest in BRCA-depleted HeLa cells. Polα inhibition by treatment with 10 μM ST1926 suppresses this defect, indicating that this PCNA retention on nascent DNA occurs at lagging strand gaps. Representative micrographs (scale bar represents 10 μm) (a) and quantifications (b) are shown. At least 50 positive cells were quantified for each condition. Center line indicates the median, bounds of box indicate the first and third quartile, and whiskers indicate the 10th and 90th percentile. The p-values (Mann–Whitney test, two-tailed) are listed at the top. A schematic representation of the SIRF assay conditions is also presented. c DNA fiber combing assay showing that co-depletion of BRD3 or BRD4 restores HU-induced fork degradation in CHAF1A-knockdown HeLa-BRCA2KO cells. The ratio of CldU to IdU tract lengths is presented, with the median values marked on the graph. The p-values (Mann–Whitney test, two-tailed) are listed at the top. A schematic representation of the DNA fiber combing assay conditions is also presented. Western blots confirming the co-depletion are shown in Supplementary Fig. 7f. d, e Chromatin fractionation experiments showing that ATAD5 (d) or LIG1 (e) knockdown increases the chromatin levels of wild-type PCNA, but not those of PCNA C81R and D150E variants. MRE11 is used as a control for the chromatin fraction. LIG1 depletion is confirmed by western blot. ATAD5 depletion is confirmed by RT-qPCR, since no antibody was available to use to verify depletion by western blot. The average of two technical replicates is shown. f DNA fiber combing assay showing that BRCA2 knockdown causes HU-induced fork degradation in 293T cells expressing wild-type PCNA, but not in 293T cells expressing PCNA variants with deficient chromatin retention. The ratio of CldU to IdU tract lengths is presented, with the median values marked on the graph. The p-values (Mann–Whitney test, two-tailed) are listed at the top. A schematic representation of the DNA fiber combing assay conditions is also presented. Western blots confirming the knockdown are shown in Supplementary Fig. 7i. g DNA fiber combing assay showing that CHAF1A knockdown suppresses HU-induced fork degradation caused by B02 treatment in 293T cells expressing wild-type PCNA, but not in 293T cells expressing PCNA variants with deficient chromatin retention. The ratio of CldU to IdU tract lengths is presented, with the median values marked on the graph. The p-values (Mann–Whitney test, two-tailed) are listed at the top. A schematic representation of the DNA fiber combing assay conditions is also presented. Western blots confirming the knockdown are shown in Supplementary Fig. 7j. h Schematic model outlining the proposed mechanism for fork degradation in BRCA-deficient cells caused by PCNA unloading defects. The failure of BRCA-deficient cells to restrain fork progression during replication stress causes gap accumulation due to repriming by PRIMPOL (on the leading strand) and Polα (on the lagging strand). Polα-mediated repriming results in formation of lagging strand gaps which preclude PCNA unloading. The persistence of PCNA behind replication forks sequesters CAF-1 away from active replication factories, thus interfering with nucleosome assembly at forks and priming them for degradation upon stalling. A more detailed version of this model figure is provided in Supplementary Fig. 8.

Fig 3: A schematic model summarizing our findings on the role of mutant FUS in inducing mitochondrial dysfunction and mtDNA instability in ALS/FTD, and its amelioration by targeted expression of DNA Lig1.The optimal FUS recruitment to mitochondria is critical for maintaining mtDNA integrity, as FUS promotes mtDNA Lig3 function through direct interaction, independent of XRCC1 in healthy neurons. However, ALS pathology associated FUS mutations lead to nuclear clearance and increased mitochondrial localization of FUS, and the mutant FUS fails to interact with mtLig3, causing defective recruitment to mtDNA damage sites and increased mutational load, ultimately resulting in mitochondrial dysfunction. The targeted expression of Lig1 in FUS-mutated cell mitochondria restores DNA repair and improves overall mitochondrial function.

Fig 4: Mitochondria DNA damage accumulation and DNA repair defects can be rescued by the correction of FUS mutations or targeted Lig1 expression.a IF of endogenous FUS localization in control and patient derived iPSC cells along with isogenic mutation corrected cells, IF was performed by anti-FUS antibody. DAPI staining indicates nucleus. Scale bar = 10 µm. b IB of endogenous FUS, HSP60, Tom20 and PCNA in FUS WT, FUS P525L and isogenic FUS L525P iPSC cells. c Human DNA ligase 1 (Lig1) localizing in mitochondria (FLAG-MTS) was generated with n-terminal FLAG and mitochondrial targeting sequence (MTS) from Cytochrome c oxidase subunit 8 (COX8) gene, immunoblot (IB) showing MTS-Lig1expression in nucleus and mitochondria in patient derived fibroblasts. d In vitro nick ligation activity assay performed using patient derived fibroblast mitochondrial extracts with MTS-Lig1 expression. e LA-PCR-based DNA damage repair kinetic analysis. Genome DNA extracted from control and patient derived fibroblasts with FUS WT, FUS P525L and FUS P525L MTS-lig1 at indicated time points after release from exposure to GO (100 ng/ml) for 1 hr. Amplification products analyzed by agarose gel electrophoresis and pico green-based quantitation represented. f microplate reader-based analysis of TMRM signal intensity, comparison of control, FUS P525L and FUS P525L cells expressing MTS-Lig1. The cells were treated with GO (100ng/ml) for 1 h and released for 2 hrs post 1 hr treatment.

Fig 5: A schematic model summarizing our findings on the role of mutant FUS in inducing mitochondrial dysfunction and mtDNA instability in ALS/FTD, and its amelioration by targeted expression of DNA Lig1.The optimal FUS recruitment to mitochondria is critical for maintaining mtDNA integrity, as FUS promotes mtDNA Lig3 function through direct interaction, independent of XRCC1 in healthy neurons. However, ALS pathology associated FUS mutations lead to nuclear clearance and increased mitochondrial localization of FUS, and the mutant FUS fails to interact with mtLig3, causing defective recruitment to mtDNA damage sites and increased mutational load, ultimately resulting in mitochondrial dysfunction. The targeted expression of Lig1 in FUS-mutated cell mitochondria restores DNA repair and improves overall mitochondrial function. Created with BioRender.com.