Fig. 5: Loss of RAD52 causes replication stress and increased DNA damage.

a Schematic representation of DNA fiber assay performed in HCT116 wild type (WT) and RAD52 knockout cells (RAD52^-/-) cells with plasmid overexpression of either RAD52^WT or RAD52^ΔC followed by incubation with 5-Chloro-2′-deoxyuridine (CldU) and 5-iodo-2′-deoxyuridine (IdU) for 30 min each to label nascent DNA. b Representative images of DNA fiber images in HCT116 WT and RAD52^-/- cells with overexpression of either RAD52^WT or RAD52^ΔC (Scale bar 2 µM). (c) Measurement of DNA fiber lengths across stated conditions described in (b) to measure replication rates. Data plotted as box and whiskers. Boxes extend from the 25th to 75th percentiles, with the median displayed as a line. The whiskers mark the minimum (1 percentile) and maximum (99th percentile). The data presented shows ≥100 DNA fibers from 3 biological replicates; p-values calculated using unpaired two tailed t-tests. d Heat map of the intensity of γH2AX ChIP signals (siNT and siRAD52 transfected HeLa cells) at genes that have a detectable R-loop peak as determined in Supplementary Fig. 6b. The γH2AX occupancy is displayed relative to the TSS ± 0.5 Mb. e Schematic representation of PLA to visualize proximity of S9.6 and γH2AX. f Representative images of the nuclear PLA foci (α-S9.6: α-γH2AX) in siNT (control), siRAD52 and siAQR transfected HeLa cells (Scale bar 10 µM). g Quantitative analysis of nuclear PLA foci across stated conditions described in (f). Data are plotted as mean ± SEM. The data presented shows ≥ 500 nuclei from 3 biological replicates; p-values calculated using unpaired two tailed t-tests.  Schematics in Fig. 5 (a) and (e) were created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license. Source data are provided as a Source Data file.