Fig 2: Generation of UFBs by HR in resolvase-deficient cells.(a) GEN1-/- cells were treated with siRNA against MUS81. They were then either untreated, or treated with Cis-Pt (1 εM for 1 h and release for 24 h), camptothecin (CPT, 1 εM for 1 h and release for 24 h) or APH (0.2 εM for 16 h). RPA2, FANCD2 and DNA were visualized as indicated. Deconvoluted images are shown. Boxed regions are enlarged and single Z planes are shown in the right.(b) Quantification of anaphase/telophase cells (30 cells per condition) with RPA2-positive UFBs, classified as with or without FANCD2 foci, as visualized in (a).(c) GEN1-/- cells were treated with siRNA against MUS81 alone or together with siRNA against BRCA2 or RAD51 for 72 h. RPA2, FANCD2 and DNA were visualized as indicated. Deconvoluted images are shown. Boxed regions are enlarged and single Z planes are shown in the right.(d) Quantification of anaphase/telophase cells (60 cells per condition) with RPA2-positive UFBs, classified as with or without FANCD2 foci, as visualized in (c).(e) GEN1-/- cells, and GEN1-/- cells stably expressing RusAWT-GEN1, were treated with siRNA against MUS81 and Cis-Pt. RPA2, RusAWT-GEN1 and DNA were visualized using anti-RPA2 antibody (red), anti-FLAG antibody (green) and DAPI (blue), respectively. Deconvoluted images are shown.(f) Quantification of cells (> 150 cells per condition) with RPA2-positive UFBs, as visualized in (e).In a, c and e, representative images of three independent experiments are shown. Quantified data in b, d and f 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 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: Unwinding of UFBs by PICH/BLM facilitates cell division.(a) 293 cells stably expressing GFP-BLMWT and GFP-BLMK695M were treated with siRNAs against GEN1, MUS81 and BLM, and Cis-Pt. RPA2, BLM and DNA were visualized using anti-RPA2 antibody (red), anti-GFP antibody (green) and DAPI (blue), respectively.(b) Quantification of cells (>100 cells per condition) with UFBs shown in (a).(c) GEN1-/- cells were treated with siRNA against MUS81 alone or together with siRNA against BLM, and Cis-Pt. RPA2, PICH and DNA were visualized using anti-RPA2 antibody (red), anti-PICH antibody (green) and DAPI (blue), respectively. Deconvulated images are shown.(d) Quantification of anaphase cells with PICH-positive UFBs (>120 cells per condition), as visualized in (c).(e) GEN1-/- cells and GEN1-/- PICH3/4 cells were treated with siRNA against MUS81 and Cis-Pt. The number of cells with RPA2-positive UFBs was quantified (>150 cells per condition).(f) GEN1-/- and GEN1-/- PICH3/4 cells were treated as shown in the scheme (upper panel) and their DNA content distributions were determined by FACS analysis (lower panel).(g) The percentage of cells with 8N DNA content, as determined in (d), was quantified.In a, c and f, representative data of three independent experiments are shown. Quantified data in b, d, e and 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. Scale bars, 10 εm.

Fig 5: BLM is required for the formation of RPA-coated UFBs.(a) U2OS cells were treated with siRNA against GEN1 and MUS81, and Cis-Pt. Cells with UFBs in different stages of mitosis were counted. BLM, RPA2 and DNA were visualized as indicated. Deconvoluted images are shown. Boxed regions are enlarged.(b) Quantification of cells with UFBs (>250 cells counted) as in (a).(c) U2OS cells were treated as in (a). PICH, RPA2 and DNA were visualized as indicated and deconvoluted images are shown. Boxed regions are enlarged.(d) Quantification of cells with UFBs (>250 cells counted) as in (c).(e) GEN1-/- cells were (>150 cells per condition) treated with siRNA against MUS81 alone, or together with the indicated siRNAs in which various nucleases/helicases were targeted, and Cis-Pt. The percentages of resolvase-deficient cells at anaphase/telophase with RPA2-positive UFBs were determined.(f) GEN1-/- cells were treated with siRNA against MUS81 alone, or together with siRNA against BLM, and Cis-Pt. RPA2 and DNA were visualized as indicated.(g) Quantification of anaphase/telophase cells (>150 cells per condition) with RPA2-positive UFBs. GEN1-/- cells were treated with siRNA against MUS81 alone, or together with siRNA against BLM or TREX1, and Cis-Pt.In a, c and f, representative images of three independent experiments are shown. Quantified data in b, d, e and 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, *P = 4 × 10-5, **P < 0.002. Scale bars, 10 εm.