Fig 2: EXO1 suppresses chromosomal instability in Nbs1 mutants. (A) Cell growth determined using a modified 3T3 protocol. Primary MEFs were counted and replated every 3 days and cumulative growth was plotted for the indicated genotypes. The genotypes are abbreviated throughout the figure as follows: wild type = W, Nbs1ΔB/ΔB = N, Exo1−/− = E, Nbs1ΔB/ΔBExo−/− = NE. (B) Premature senescence in NE double mutant cultures. Passage 6 MEFs were stained for senescence associated (SA) β-galactosidase activity and counted under magnification. The percentage of SA β-gal positive cells is plotted. (C) DNA synthesis is reduced in NE cell cultures. Passage 2 MEFs were pulsed with BrdU for 4 h and the percentage of BrdU positive cells was measured by flow cytometry combined with propidium iodide (PI) staining. (D) Increased anaphase bridges and micronuclei are evident in NE cultures. Quantification of the percentage of aberrant mitoses in primary cell cultures. A minimum of 100 mitoses are scored in each case. (E) Increased chromosomal instability in primary, early passage (p2-p3) NE cell cultures. The number of metaphase aberrations per metaphase were scored and binned as indicated in the figure. A higher percentage of NE metaphases show more than two aberrations per metaphase. (F) Plot of chromosomal aberration types (cd.=chromatid break, chr.=chromosome break, fus.=fusion, frag.=fragment) scored from multiple primary cell cultures depicted as percent aberrations per chromosome. NE cultures have particularly high numbers of chromatid breaks. Examples of aberrations scored in a partial NE metaphase are shown in the right panel. Red arrows indicate cd aberrations and blue arrow indicates a fusion. Additional examples are provided in Supplementary Figure S1. (G) Plot of chromosomal aberration types (as in (F)) identified in transformed cell cultures of the indicated genotypes. Total aberrations remain higher in NE cultures although chromosome fragments were the predominant lesion identified.

Fig 3: Differential effects of EXO1 loss on the damage sensitivity of Nbs1 mutants. Sensitivity of the cell cultures of the indicated genotypes to DNA damaging agents or PARP inhibitor (Olaparib) using the clonogenic survival assay. The genotypes are abbreviated in each case as follows: wild type = W, Nbs1ΔB/ΔB = N, Exo1−/− = E, and Nbs1ΔB/ΔB Exo−/− = NE. (A) IR sensitivity following treatment with 2, 5 or 8 Gy. (B) Cisplatin sensitivity following treatment at the indicated doses for 2 h. (C) UVC sensitivity after treatment with 15 or 25 J/m2. (D) Olaparib sensitivity following continuous treatment with the indicated doses. (E) CPT sensitivity (high) following treatment with the indicated doses for 1 h. (F) CPT sensitivity (low) following treatment with the indicated doses for 24 hours.

Fig 4: EXO1 influences DNA repair and DNA replication in Nbs1 mutants. (A) Schematic illustration of the SA-GFP based SSA assay. (B) Western blotting of cells transfected with or without a vector expressing human NBS1. The genotypes are abbreviated throughout the figure as follows: wild type = W, Nbs1ΔB/ΔB = N, Exo1−/− = E, and Nbs1ΔB/ΔB Exo−/− = NE. (C) Quantification of SSA mediated repair plotted as the percentage of GFP positive cells. Values for NE are corrected for the reduced percentage of cells in S/G2 determined by BrdU and PI staining (mean 58% compared to a mean of 73% in the other genotypes). The fold rescue with NBS1 expression is the same in N and NE (2.2 fold). (D) Measurement of replication tract lengths following CldU or IdU pulse labeling. (E) Calculation of replication fork velocity in the indicated genotypes (as described in ‘Materials and Methods’ section). Examples of representative forks from W and NE cultures are shown for comparison. (F) Assessment of replication fork restart following 1 mM HU treatment using the indicated scheme. Relative tract ratio is calculated by dividing the length of the IdU tract by that of the CldU tract ( = 2 under unperturbed conditions). Thus, higher values indicate faster restart following HU removal.

Fig 5: UFL1 protein accumulates at DSBs through the MRN complex. a U2OS cells stably expressing UFL1 Tet-on shRNA were treated with doxycycline (Dox) for 3–5 days, and then treated with or without 2 Gy IR. After 30 min, cells were harvested and lysed with NETN buffer. Cell lysates were incubated with UFL1 antibody + Benzonase. The immunoprecipitates were blotted with indicated antibodies. b Immunofluorescence of UFL1 and γH2AX in U2OS cells irradiated with IR (0.5 Gy). c Triamcinolone acetonide (TA) treatment induces the translocation of RFP-I-SceI-GR fusion protein from the cytoplasm to the nucleus and generates one double strand break at the cutting site. The protein localization was detected by indicated antibodies. d UFL1 foci formation at different time points following 0.5 Gy IR treatment. e, f UFL1 foci formation was analyzed in Mre11 knockdown cells or NBS1 deficient cells (NBST). g NBST cells were transfected with Vector (V) or Flag-NBS1 and treated with or without 2 Gy IR. After 30 min, the cells were lysed and immunoprecipitation with UFL1 antibody with Benzonase treatment was performed. The immunoprecipitates were blotted with indicated antibodies. h The schematic diagram of NBS1 protein domain. i U2OS cells were treated with or without 2 Gy IR, and the cell lysates were pulled down with GST, GST-NBS1 FHA+BRCT (1+2) proteins. After washes, the beads were boiled and analyzed with indicated antibodies. Scale bars, 10 µm. Source data are provided as a Source Data file