Fig. 1: RAD52 association with the transcriptional complex.
From: RAD52 resolves transcription-replication conflicts to mitigate R-loop induced genome instability
a Schematic representation of the workflow for the identification of RAD52 interacting proteins. HA-control and HA-RAD52 immunoprecipitation was performed in HEK293T cells using α-HA tagged magnetic beads for the pulldown followed by Mass spectrometry (MS). b Volcano plot of the proteins identified in RAD52 IP-MS in n = 3 biologically independent experiments. Mean log2 fold change in protein intensities on the x-axis of all replicates between HA and HA-RAD52 are plotted against the −log10 adjusted p-value (Student’s two-sided t-test with equal variance) on the y-axis. 212 proteins were identified to be significantly enriched. Significantly enriched proteins in blue (p < 0.05) and non-significant in grey. c Co-immunoprecipitation of endogenous RAD52 binding proteins in HeLa cells. RAD52 and IgG antibodies were used to immuno-precipitate proteins and analyzed by immunoblotting with indicated antibodies. Results reproducible for at least 2 biological replicates. d Schematic representation of PLA to visualize proximity of RAD52 protein and RNA Pol II. e Representative images of the nuclear PLA foci (α-RAD52: α-RNA Pol II S2) across stated conditions (Scale bar 10 µM). f Quantitative analysis of nuclear PLA foci from (e) 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. g Metagene plots showing the distribution of the RNA Pol II and RAD52 Chromatin immunoprecipitation sequencing (ChIP-seq) peaks (IP/input) in HeLa cells across genes and the flanking regions ( ± 10 kb). TSS: Transcription Start Site, TES: Transcription End Site. h Heatmap representing RNA Pol II and RAD52 ChIP-seq tracks, centered at the TSS and TES ± 10 kb, and rank-ordered according to RNA Pol II occupancy. i Bar chart showing how RNA Pol II and RAD52 peaks are distributed across different genomic regions as indicated. Peaks were obtained with MACS2. Genome wide distribution is shown on top for comparison. j Venn diagram showing the overlap of peaks RNA Pol II ChIP and RAD52 ChIP according to MACS2 across the genome. k A representative snapshot of chromosome 19 depicting RNA Pol II (red) and RAD52 (green) ChIP binding sites in control HeLa cells. Input DNA (grey) represents a negative control for background normalization. Schematics in Fig. 1 (a) and (d) 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.
