Fig 2: TRAIP is required to process sites of incomplete DNA replication and preserve genome integrity in response to DNA replication stress in human cells.(A) U2OS control cells and those expressing wild type TRAIP were treated as described in Figure 3B, except that cells were re-seeded onto plates for 15 min after mitotic shake-off, before detection of DAPI-positive DNA bridges in anaphase cells (white arrow denotes bridge - the data are quantified in the right-hand panel). (B) As per (A), except that ultra-fine DNA bridges were monitored by immunofluorescence detection of the PICH ATPase. Chromosomal DNA was detected by DAPI-staining. Note that the ultra-fine bridges are DAPI-negative. A quantification of the data is presented in the right-hand panel. (C) An analogous experiment to those above was performed, except that cells were re-seeded for 2.5 hr before detection of the 53BP1 protein by immunofluorescence in newly-born G1 cells. The number of 53BP1 bodies per G1-phase cell is quantified in the right-hand panel. (D) As per (C), except the number of micronuclei per G1-phase cell (indicated by white arrows) was determined by DAPI-staining. Statistics presented in this figure were calculated as described above for Figure 3. The scale bars correspond to 2 µm.

Fig 3: Interaction of BRCA1 and 53BP1 in the tolerance to cytotoxic agents.a–f Cytotoxicity measurements on DT40 cell lines of the indicated genotypes using drugs named above each panel. Results are shown as mean and SEM of relative IC50 concentrations compared to the WT cell lines, individual values derived from n = 3 independent experiments are indicated. The mean IC50 value of the WT cell line is displayed on each panel. Source data are provided as a Source Data file.

Fig 4: 53BP1 suppresses gene conversion in the absence of BRCA1.a Schematic map of the rearranged chicken immunoglobulin VL1 gene. The coding region is shown in grey, darker grey boxes indicate the hypervariable complementarity-determining regions (CDR). Primer pairs for an initial genomic PCR reaction (CVLF5 and CVLR3) and a subsequent nested indexed PCR (VL-1F and VL-2R) are shown. b SBS and gene conversion events detected in cell populations derived from individual DT40 cell clones of the indicated genotypes that were cultured for 40 days, presented as the number of mutation events per total number of sequenced read pairs in the same sample. Detected point mutation (SBS) events at AT or CG base pairs are shown separately. Bars show means, individual values of 3 parallel cell clones are indicated with black markers. The significance of tested differences is shown (unpaired two-sided t-test, ns not significant). c The maximum possible length of each individual gene conversion tract in sequence reads derived from cell populations of the indicated genotypes. The median, upper and lower quartiles are shown, the indicated statistical differences were calculated with the Wilcoxon rank-sum test. Source data are provided as a Source Data file.

Fig 5: Influence of PNKP variants on cellular response to radiation and DNA repair. (A) Viability of wild-type and mutant cell lines at different radiation dosages. Plots represent mean \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\pm \hspace{0.17em}$$\end{document}±SD. Ordinary one-way ANOVA was performed to compare each cell line with the wild-type HeLa at the same radiation dose using GraphPad Prism 7.0. (B) Single-strand break repair measured by the single cell gel electrophoresis assay. DNA tail moments of cell lines were measured at 1 and 24 h after irradiation. Each column includes data from at least 300 cells. Plots represent mean ± SEM. At each time point, one-way ANOVA was performed to compare each group with the wild-type un-transfected HeLa cells at the same time point using GraphPad Prism 7.0. (C) Repair of DNA double strand breaks by the NHEJ pathway based on 53BP1 foci numbers observed in the wild-type and stably transfected cell lines at different times after irradiation, n indicates the measured cell numbers in each group. The plots represent mean ± SD. In each graph, one-way ANOVA was performed to compare 53BP1 foci at each time point with the number of foci in unirradiated cells using GraphPad Prism 7.0, GraphPad Software. ***p < 0.001, ****p < 0.0001.