Fig 2: MDC1 is dispensable for NBS1’s function at meiotic prophase.a Immunofluorescence staining analyses of MDC1 and NBS1 in Mdc1+/+ and Mdc1-/- MEFs after treatments of 10 µM bleomycin for 6 h or 5 µM etoposide for 2 h. ?H2AX marks DNA damage sites. Scale bar, 10 µm. b Immunofluorescence staining of SYCP3 and ?H2AX were used for analyses of stages of meiotic prophase in spermatocytes from Mdc1-/- mice. Scale bar, 5 µm. Surface spreads of zygotene spermatocytes from Mdc1+/+ and Mdc1-/- mice were incubated with antibodies against RAD51 (c), DMC1 (d), RPA2 (e), and MEIOB (f). Scale bar, 5 µm. g Localization of MDC1 in surface spreads of pachytene spermatocytes from Mdc1+/+ and Mdc1-/- mice. Scale bar, 5 µm. h Localization of endogenous NBS1 in surface spreads of pachytene spermatocytes from Mdc1+/+ and Mdc1-/- mice. Scale bar, 5 µm. Cartoons depicting proteins (red) localized on chromosome loops and axes (green) are shown on the right. Arrows mark chromosome axes and arrowheads mark chromosome loops. i Localization of endogenous NBS1 in surface spreads of pachytene spermatocytes from H2ax+/+ and H2ax-/- mice. Scale bar, 5 µm. Cartoons depicting proteins (red) localized on chromosome loops and axes (green) are shown on the right. Arrows mark chromosome axes and arrowheads mark chromosome loops.

Fig 3: RNA-Seq analysis revealed transcriptional alteration in fibroblasts, iPSCs and NPCs. (A) Fibroblasts (NB1RGB), iPSCs (NB1RGB C2 and 201B7) and NPCs (C2) were irradiated at 5 Gy. One hour after IR, RNA samples were extracted and analyzed using the Illumina HiSeq 2500 next-generation sequencer for RNA-Seq. Raw data were quality controlled and aligned to the reference hg19. Heat maps classed as HR repair, NHEJ repair and apoptosis were obtained using R software. Red, upregulated; green, downregulated. (B) Differential expression patterns of each gene were represented by FPKM. Expression differences were classified according to molecular machinery components: DNA repair, cell cycle checkpoints and apoptosis. (C) Differential expression patterns by RNA-Seq were confirmed by immunoblotting. ATM, NBS1, CHK1, p53 and p21 antibodies were used for immunoblotting. GAPDH antibody was used as loading control. Immunoblotting data were obtained using C-digit as digital data. Full scan data of each band are provided in Supplementary Figs 3 and 4. RNA-Seq = RNA sequencing, iPSCs = induced pluripotent stem cells, NPCs = neural progenitor cells, HR = homologous recombination, NHEJ = nonhomologous end joining, FPKM = fragments per kilobase of exon per million reads mapped.

Fig 4: NBS1 is required for chromosome synapsis and meiotic progression.a Immunofluorescence staining of SYCP3 and ?H2AX was used for analyses of stages of meiotic prophase in spermatocytes from control and Nbs1 vKO mice. Scale bar, 5 µm. b Percentage of spermatocytes at each stage of meiotic prophase from control and Nbs1 vKO mice. Error bars represent standard deviation, n = 3. L leptotene, Z zygotene, P pachytene, D diplotene. c Typical abnormal chromosome structures in Nbs1 vKO zygotene spermatocytes. White boxes indicate the sites of abnormal chromosomes, and are magnified on the right. Scale bar, 5 µm. d Number of abnormal chromosome structures per zygotene spermatocyte from control and Nbs1 vKO mice. Error bars represent SEM, n = 30. e Immunofluorescence staining of SYCP1 and SYCP3 were used for analyses of chromosome synapsis at late leptotene and zygotene stages of meiotic prophase from control and Nbs1 vKO mice. Scale bar, 5 µm. Defective chromosome synapsis was observed in all Nbs1 vKO spermatocytes (100%) but none of the control spermatocytes (0%).

Fig 5: MRE11 SUMOylation is increased in response to DNA damage.a, b HeLa cells were treated with or without 20 µM ML792 for 1 h and the indicated times of 1 µM CPT (a), or the indicated doses of ML792 and 1 µM CPT for 1 h (b) and the whole cell extracts were subjected to immunoblotting. c, d ML792 repressed pRPA2 (S4/S8) foci formation in S/G2 phase HeLa cells after 1 µM CPT treatment for 45 min. Scale bar, 10 µm. The data are presented as means ± SD, n (DMSO) = 117 cells, n (ML792) = 119 cells. e MRE11 was modified by SUMO1/2/3. HEK293T cells were cotransfected with SFB-MRE11 and 10×His-SUMO1/2/3, followed by Ni-NTA pull-down with guanidine denaturing buffer. f NBS1 SUMOylation was tested as in e. g MRE11 SUMOylation was enhanced by treatment with DSB-inducing agents (1 µM CPT for 2 h, 100 µM etoposide for 3 h, and 1 µM cisplatin for 3 h) but not HU (2 mM, 3 h). h MRE11 SUMOylation increased after 1 µM CPT treatment in a time-dependent manner. i NBS1 SUMOylation was downregulated upon treatment with DNA-damaging agents as in g. j NBS1 SUMOylation gradually decreased along CPT treatment. k The indicated purified proteins were analyzed by Coomassie blue staining, and then used in reconstituted reactions to test MRE11 SUMOylation in vitro. l SUMOylation of endogenous MRE11 was examined in HEK293T cells treated with or without CPT (1 µM, 6 h). m HEK293T cells expressing SFB-MRE11 and His-SUMO2 were fractionated into soluble and chromatin fractions, followed by the analysis of MRE11 SUMOylation.