Fig. 2: RAD51 association with mitotic chromatin promotes MiDAS.

a Schematic diagram of experimental procedure in which cells were synchronised by double thymidine block and release (dT-B/R) and G2/M arrest by RO3306 treatment, and exposed to small molecule inhibitors of RAD51 (B02) or vehicle (-) upon mitotic release (m-R). Subsequently, mitotic cells were collected by mitotic shake-off (m-SO). b Depiction of RAD51 association with resected ssDNA and associated γ-H2AX, spanning an estimated 1–2 Mb. B02 blocks de novo RAD51 loading but does not disrupt a pre-formed filament. c, d Mitotic cells collected by m-SO, following 10 µM EdU exposure with or without B02 at the indicated concentrations, were stained and quantified for EdU foci (c) or γ-H2AX foci (d) per mitotic cell. Data were obtained from three independent experiments (100 cells analysed per repeat); n = 300 per condition. Data distribution is represented by Tukey box-and-whisker plots. Bounds of box are 25–75th percentile, centre shows the median and ‘+’ marks the mean. Whiskers indicate ±1.5xIQR, data outside this range are drawn as individual dots. p-values were calculated by a Kruskal–Wallis test followed by Dunn’s multiple comparison test. Asterisks indicate **p-value ≤ 0.01; ***p-value ≤ 0.001; ****p-value ≤ 0.0001. e Schematic diagram of experimental procedure to synchronise U2OS Flp-In T-REx cell lines expressing FLAG-tagged RAD51 variants from the tet-O promoter. Cells were synchronised in the presence of low-dose APH, with or without the addition of doxycycline as indicated for each cell line. f Western blot analysis of Lamin A (loading control) and RAD51 protein levels for the total cell population synchronised according to the workflow depicted in e. Based on a single experiment. g FACS analysis of mitotic index (MI) (pS10-H3) and DNA content (PI) following cell synchronisation according to the experimental procedure depicted in Fig. 2e. At 30 min after m-R, the total cell population was collected by trypsinization and analysed by FACS. The G1, S and G2 populations were estimated by the Watson Pragmatic algorithm (FlowJo), based on the PI staining of interphase cells. h Representative images of mitotic cells, stained for EdU and γ-H2AX, quantified in (i) and Supplementary Fig. 2e and f. Scale bar indicates 5 µm. i Quantification of the number of EdU foci that colocalise with γ-H2AX (γ-H2AX ⁺) and the number of EdU foci that do not colocalise with γ-H2AX (γ-H2AX^-) per mitotic cell in doxycycline treated cells. Data were obtained from four independent experiments; n = 400 per condition. Data distribution is represented by Tukey box-and-whisker plots. Bounds of box are 25–75th percentile, centre shows the median and ‘+’ marks the mean. Whiskers indicate ±1.5xIQR, data outside this range are drawn as individual dots. p-values were calculated by a Kruskal–Wallis test followed by Dunn’s multiple comparison test. Asterisks indicate **p-value ≤ 0.01. Source data are provided as a Source Data file.