Fig 2: Rescue of BLM/MUS81 defects by expression of GEN1nuc.(a) Clonogenic cell survival assays were carried out on Flp-In T-REx 293 cells expressing GEN1WT or GEN1nuc (±Tet) and depleted for the indicated proteins. Cell survival after treatment with the control siRNA is defined as 100%. The data represent the mean±s.d. of at least three independent experiments. P values were determined using a two-tailed t-test. (b) Representative images of metaphase spreads of Flp-In T-REx 293 cells treated with the indicated siRNAs. Three types of chromosome aberrations (chromatid breaks, isochromatid breaks and radial chromosomes) were identified and quantified. (c) Flp-In T-REx 293 cells expressing GEN1WT or GEN1nuc (±Tet) were treated with the indicated siRNAs, and metaphase spreads were analysed and quantified for the indicated chromosome aberrations. One-hundred twenty metaphase cells (>5,500 chromosomes) were analysed. The data represent the mean number of aberrations per 100 chromosomes per spread±s.e.m. P values were determined using a two-tailed t-test.

Fig 3: Persistent recombination intermediates lead to the formation of HR-UFBs.(a) 293 and GEN1-/- cells were treated with control siRNA or siRNA against MUS81 and Cis-Pt. 293 cells treated with aphidicolin (APH, 0.2 εM for 16 h) were used as controls. RPA2, FANCD2 and DNA were visualized using anti-RPA2 antibody (red), anti-FANCD2 antibody (green) and DAPI (blue), respectively. Deconvoluted images are shown. Boxed regions are enlarged and single Z planes are shown in the right.(b) Quantification of anaphase/telophase cells with RPA2-positive UFBs (150 cells per condition), with or without FANCD2 foci, as visualized in (a).(c) Cells were treated as in (a), and FANCD2 and DNA were visualized using anti-FANCD2 antibody (green) and DAPI (blue), respectively. Deconvoluted images are shown.(d) Quantification of prometaphase cells with >4 FANCD2 twin foci (150 cells per condition), as visualized in (c).(e) 293 cells and GEN1-/- cells were treated with control siRNA or siRNA against MUS81 for 48 h, and then labelled with CldU and IdU for DNA fibre analysis. Representative fibres are shown.(f) Quantification of IdU track length relative to 293 control cells (>200 fibres per condition), as in (e).(g) Representative images of hTERT-RPE1 cells treated with siRNAs against GEN1 and MUS81, and Cis-Pt. RPA2, FANCD2 and DNA were visualized using anti-RPA2 antibody (red), anti-FANCD2 antibody (green) and DAPI (blue), respectively.(h) Quantification of anaphase/telophase hTERT-RPE1 cells treated with control siRNA, siRNA against GEN1 and/or MUS81, and Cis-Pt. Cells with RPA2-UFBs were classified as with or without FANCD2 foci (>100 cells per condition).In a, c, e and g, representative images of three independent experiments are shown. Quantified data in b, d, f and h represent the mean ± s.d. of n = 3 independent experiments. Source data are available in Supplementary Table 1. P values were determined using a two-tailed t-test. Scale bars, 10 μm.

Fig 4: HR-UFB breakage leads to DNA damage and gross chromosome abnormalities.(a) 293 cells, and GEN1-/- cells, were treated with control siRNA or siRNA against MUS81 and Cis-Pt, and released into fresh media with or without nocodazole (Noc, 100 ng/mL) and reversine (Rev, 0.5 eM) for 24 h. MDC1, Cyclin A, and DNA were visualized using anti-MDC1 antibody (green), anti-cyclin A antibody (red), and DAPI (blue), respectively. Scale bar, 10 µm.(b) Quantification of G1 cells (>400 cells per condition) with >4 MDC1 foci, as in (a).(c) 293 cells and GEN1-/- cells were treated with control siRNA or siRNA against MUS81, followed by Cis-Pt (1 eg/mL) and BrdU (10 eM) treatment for 1 h. Cells were released into fresh media, collected at the indicated time points and analysed by FACS. The DNA content histograms of BrdU-negative mock-depleted 293 cells, MUS81-depleted 293 cells, mock-depleted GEN1-/- cells and MUS81-depleted GEN1-/- cells, at the indicated time points are shown.(d) Representative images of metaphase spreads from GEN1-/- cells treated with siRNA against MUS81 and Cis-Pt, and released into fresh media for 48 h. Representative end-to-end fusions and radials are shown.(e) 293 and GEN1-/- cells were treated as in (d), and 75 metaphase spreads per condition were analysed for chromosome fusions in three independent experiments.(f) GEN1-/- cells were treated as in (d), except that DMSO or the DNA-PK inhibitor NU7026 (10 eM) were added 24 h before harvest. Control siRNA-treated 293 cells were used as control. 60 metaphase spreads per condition were analysed for chromosome fusions.(g) Cells were treated as in (f) except Noc and Rev were added to the cells instead of NU7026. 45 metaphase spreads per condition were analysed in three independent experiments.In a and d, representative images of three independent experiments are shown. Quantified data in b and e-g represent the mean ± s.d. of n = 3 independent experiments. Source data are available in Supplementary Table 1. P values were determined using a two-tailed t-test.

Fig 5: General mechanism for the formation of single-stranded UFBs.(a) 293 cells were treated control siRNA or siRNA against BLM, and ICRF-193 (0.1 εM, 1 h). PICH, RPA2 and DNA were visualized as indicated.(b) Quantification of cells (>100 cells per condition) with RPA2-coated UFBs in (a).(c) GEN1-/- cells and GEN1-/- PICH3/4 cells were treated with ICRF-193 (0.1 εM, 1 h). PICH, RPA2 and DNA were visualized.(d) Quantification of cells (>120 cells per condition) with RPA2-coated UFBs in (c).(e) Schematic diagram of the four types of anaphase UFB: 1) Centromere-UFBs (C-UFBs) emerge from centromeric regions, possess double-stranded catenanes and are resolved by PICH/BLM and topoisomerase II. Recruitment of BLM is required for RPA formation; 2) Fragile Site UFBs (FS-UFBs) emerge from incompletely replicated DNA at CFSs (yellow rhombus) and are flanked by FANCD2 twin foci. The bridges frequently possess regions that are bound by RPA; 3) Telomere-UFBs (T-UFBs) originate from telomere fusions, persist and develop into chromatin bridges that are processed by TREX1 to ultimately generate ssDNA; 4) Homologous recombination-UFBs (HR-UFBs) originate from unresolved recombination intermediates. In the absence of GEN1/MUS81, HR-UFBs accumulate at anaphase and PICH/BLM are recruited for DNA unwinding, ssDNA formation and breakage at mitosis. See text for further details.In a and c, representative images of three independent experiments are shown. Quantified data in b and d represent the mean ± s.d. of n = 3 independent experiments. Source data are available in Supplementary Table 1. P values were determined using a two-tailed t-test. Scale bars, 10 εm.