Fig. 4: RAD52 recruits TOP2A to mitigate transcription-replication conflicts.
From: RAD52 resolves transcription-replication conflicts to mitigate R-loop induced genome instability
a Schematic representation of PLA to visualize proximity of RAD52 and TOP2A. b Representative images of the nuclear PLA foci (α-RAD52: α-TOP2A) in siNT (control) and siAQR transfected HeLa cells (Scale bar 10 µM). c Quantitative analysis of nuclear PLA foci across stated conditions described in (b). The data presented shows ≥ 500 nuclei from 3 biological replicates. d Schematic representation of PLA to visualize proximity of HA-tagged RAD52 (HA-RAD52) and TOP2A. e Representative images of the nuclear PLA foci (α-HA: α-TOP2A) in siRAD52 (5’UTR) transfected HeLa cells with overexpression of either RAD52WT or RAD52ΔC (Scale bar 10 µM). f Quantitative analysis of nuclear PLA foci across stated conditions described in (e). The data presented shows ≥ 500 nuclei from 3 biological replicates. g Representative images of S9.6 immunostaining to detect R-loops in siNT (control) and siTOP2A transfected HeLa cells. RNase H treatment was added as a negative control to eliminate R-loops (Scale bar 10 µM). h Quantitative analysis of nuclear S9.6 foci across stated conditions from (g). 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 ≥ 500 nuclei from 3 biological replicates; p-values calculated using unpaired two tailed t-tests. i Schematic representation of PLA to visualize proximity of PCNA and RNA Pol II to measure TRCs. j Representative images of the nuclear PLA foci (PCNA: RNA Pol II S2) in siNT (control) and siTOP2A transfected HeLa cells (Scale bar 10 µM). k Quantitative analysis of nuclear PLA foci across stated conditions described in (j). The data presented shows ≥ 500 nuclei from 3 biological replicates (l) Schematic representation of PLA to visualize proximity of S9.6 and TOP2A. m Representative images of the nuclear PLA foci (α-S9.6: α-TOP2A) in siNT (control), siRAD52 and siAQR transfected HeLa cells (Scale bar 10 µM). n Quantitative analysis of nuclear PLA foci across stated conditions described in (m) normalized to siNT. The data presented shows ≥ 500 nuclei from 3 biological replicates. o Mechanistic model of RAD52 role in preventing transcription-replication conflicts. In Fig. 4 (c–k) and (n), data are plotted as mean ± SEM and p-values calculated using unpaired two tailed t-tests. Schematics in Fig. 4 (a) (d) (i) (l) and (o) 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.
