Fig 2: SUV39h1/2 compacts chromatin and antagonizes BRCA1-dependent resection to promote NHEJ.a, b Recruitment of SUV39h1 (a) to laser-induced DNA damage sites in wild-type, 53BP1?, RIF1? or ASF1a?ASF1bH HEK293T cells and quantification (b). The mean and s.e.m. values are shown for every time point. c Immunoblots showing the knockdown efficiency of SUV39h1/2 in U2OS-265 cells. d–h GFP-HP1? (d), H3K9me3 (e), BRCA1 (f), and RPA32 (g) signals in the array and quantification (h) in SUV39h1/2-depleted U2OS-265 cells after DSB induction. The mCherry-LacR area was also quantified in (h). For BRCA1 and RPA32 staining, cells were synchronized into the G1 phase as described previously91. Some experiments were carried out together with those in Fig. 5b, c and thus the same controls were used. The error bars represent the s.e.m. The numbers of cells pooled from two independent experiments are indicated. i, j Etoposide-sensitivity assay (i) and random integration assay (j) of wild-type and SUV39h1/2 double-knockout DT40 cells. The mean and s.d. of the results from three independent experiments are shown. k Olaparib-sensitivity assay of brca1-/-, suv39h1/2dko and suv39h1/2dkobrca1-/- DT40 cells. The mean and s.d. of the results from three independent experiments are shown. *P < 0.05; ***P < 0.001; ****P < 0.0001. Statistical analysis was performed using the two-tailed t test. Uncropped images of the gels and statistical source data including the precise P values are provided in Source data.

Fig 3: P1’s Cells Exhibit a Reduced DNA End Resection and HDR(A–C) Immunofluorescence detection of P-KAP1 (A), RPA32 (B), and RAD51 (C) in primary fibroblasts from healthy control and P1 before and 6 h after 10 Gy irradiation. The image is representative of three independent experiments.(D) Quantitative analysis of RAD51 foci before and after IR in SV40-hTERT fibroblasts untransduced or transduced with an empty or WT-RAD50-expressing vector. The number of nuclei scored and statistical significances are noted.(E) Efficiency of HDR assessed by the Cas9-directed knockin of Clover in the Lamin-A (LMNA) coding sequence. Cells were transfected with the mCherry vector alone (used to control transfection efficiency) or in combination with the repair template sequence containing the Clover coding sequence in conjunction, or not, with the pX330-LMNA1 vector encoding Cas9 and the guide RNAs (gRNAs) targeting the LMNA sequence (denoted as CRISPR-Cas9). Relative HDR efficiency was measured by the percentage of Clover+ cells triple transfected relative to cells transfected without the CRISPR-Cas9-gRNA-expressing vector. Results represents the mean and SD of triplicates from three independent experiments.

Fig 4: Typhoid toxin exhausts RPA by oversupply of ssDNA substrate. a Representative image of toxin-induced RPA exhaustion. HT1080 cells intoxicated with toxinWT or toxinHQ for 2 h before polymerisation of double-stranded BrdU-labelled DNA from sites of exposed ssDNA template at 24 h (ssDNA, magenta). ssDNA-bound RPA32 pT21 (pRPA, cyan) shown with BrdU shown in grayscale for clarity (ssDNA foci). Insets of toxinWT magnify ssDNA foci unprotected by pRPA (blue arrow) or partially protected by pRPA indicated by white co-localisation staining (white arrow). Insets of toxinHQ show RPA-bound ssDNA. Scale bars 10 µm. b Proportion of BrdU-labelled ssDNA foci per nucleus in intoxicated HT1080 cells. Aphidicolin (APH) was used as positive control for ssDNA induction and toxinHQ used as a negative control. Coloured circles indicate means from fields of view. Two biological replicates (160 nuclei per variable). Error bars SEM. c Proportion of serum-starved cells with RPA32 pT21 foci (pRPA). Coloured circles indicate means from technical replicates (two biological replicates, 900 nuclei/variable). Error bars SEM. d Proportion of cells with RPA32 pT21 foci (pRPA) in presence of serum. Coloured circles indicate means from technical replicates (2 biological replicates, 900 nuclei/variable). Error bars SD. e Representative image of RPA32 pT21 (pRPA, grayscale) with ?H2AX (magenta) RINGs (blue arrows) in cells treated with APH as indicated. Scale bars 50 µm. f Proportion of ?H2AX RINGs in APH-treated cells ± toxinWT as indicated. Coloured circles indicate means from fields of view (two biological replicates, 600 nuclei/variable). Error bars SEM. g Mean RPA pT21 (pRPA) foci intensity in APH-treated cells ± toxinWT as indicated. Coloured circles indicate means from fields of view (two biological replicates, 600 nuclei/variable). Error bars SEM. A.U, arbitrary units. In representative images (a, e) outlines of DAPI-stained nuclei are shown. Statistical significance (**** = P < 0.0001, *** = P < 0.0002, ** = P < 0.0021, * = P < 0.0332, ns = P > = 0.05) was calculated relative to toxinHQ using one-way ANOVA and a Dunnett’s multiple comparison test (b), or relative to untreated via one-way ANOVA and a Tukey’s multiple comparison test (c, d, f, g). Source data provided as a Source Data file

Fig 5: Effects of RFWD3 knockdown on RPA ubiquitination after illudin S treatment and effects of ICL-repair factors deficiency or knockdown on illudin S or irofulven survival and DNA synthesis.(A) WI38VA13 cells were transfected with siNTC#2 or siRFWD3#1 and cultured for 2 d. Cells were then transfected with siRNAs and the FLAG-ubiquitin expression plasmid. 24 h after transfection, cell extracts were prepared and immunoprecipitated using anti-FLAG. Precipitated proteins were analyzed by immunoblotting with anti-RPA2, anti-RAD51, and anti-FLAG antibodies. (B) WI38VA13[WT] and [KR] cells were transfected with the FLAG-ubiquitin expression plasmid. Immunoprecipitation and immunoblotting were performed as in (A). (C) BRCA1-deficient HCC1937 cells and BRCA1-expressing HCC1937 (HCC1937+BRCA1) cells were exposed to the indicated doses of illudin S for 4 d. Cellular survival was evaluated by MTS assay. Data are represented as means ± SD of n = 4 independent experiments. (D) Whole-cell lysates were prepared from the cells used in (C) and analyzed by immunoblotting with anti-BRCA1 and anti-Lamin B antibodies. (E) FANCD2-deficient PD20F, PD20F/vector, and PD20F/3xFLAG-FANCD2 cells were exposed to illudin S for 4 d and cellular survival was evaluated by MTS assay. Data are represented as means ± SD of n = 4 independent experiments. (F) Whole-cell lysates were prepared from the cells used in (E) and analyzed by immunoblotting using anti-FANCD2 and anti-Lamin B antibodies. (G, H) PD20F and PD20F/3xFLAG-FANCD2 cells were exposed to illudin S for 1 h (G) or irofulven for 24 h (H) and cultured without the drug until colonies formed. Data are represented as means ± SD of n = 3 independent experiments. (I) WI38VA13 siRNA against FANCD2 (siFANCD2) or BRCA1 (siBRCA1#2). Transfected cells were treated with irofulven for 1 h. After 4 d, cellular survival was evaluated by MTS assay. Data are represented as means ± SD of n = 3 independent experiments. (J) Whole-cell lysates were prepared from the cells used in (I) and analyzed by immunoblotting with anti-BRCA1, anti-FANCD2, and anti-Lamin B antibodies. (K) siNTC#3, siRFWD3#2, siRAD51, or siBRCA1#2 transfected cells were subjected to DNA fiber assay as described in Fig 3K. Quantified CldU and IdU track length. At least 100 tracks from two independent experiments were evaluated. The line represents the median; boxes are the 25th and 75th percentiles; whiskers are the minimum and the maximum. Statistical significance was evaluated by unpaired t test (two-tailed). (L) Whole-cell lysates were prepared from the cells used in (K) and analyzed by immunoblotting with anti-RFWD3, anti-BRCA1, anti-RAD51, and anti-Lamin B antibodies.Source data are available for this figure.