Extended Data Fig. 8: Characterization of seDSB damage, DDR and initial resection.
From: BRCA2 prevents PARPi-mediated PARP1 retention to protect RAD51 filaments
(a) U2OS NT and 0.1 µM CPT treated cells were pulse-labeled with 10 µM EdU for 30 min and analyzed by flow cytometry for DNA synthesis (EdU) and DAPI staining for DNA content (10,000 events per sample) (r = 2). (b) Experimental schematic of inducing seDSB repair foci and EdU labeling strategy to visualize nascent DNA (naDNA). (Left) Levels of γH2AX (n = 95) and 53BP1 (n = 82) localizing at nascent DNA 1 h after CPT treatment. Individual data points represent result from single cell (r = 2). (Right) Representative super-resolution images of a single nucleus, post damage, and stained for naDNA (yellow) and 53BP1 (magenta). Scale bar = 2 μm. (c) Western blot analysis for DNA Damage Response (DDR) pathway of U2OS cells treated with 0.1 µM CPT for 1 h. (d) Experimental schematic depicting spatiotemporal mapping of the arrivals, accumulations, and departures of MRE11(n1Hr = 248; n3Hrs = 234) and RPA (n1Hr = 363; n3Hrs = 238) at individual seDSBs. (e-f) Kinetics of MRE11 and RPA localizing at nascent DNA throughout repair over 3 h recovery (r = 2). (g) Representative super-resolution images of a single nucleus, post damage, and stained for naDNA (yellow), MRE11 (green) and RPA (red). Scale bar = 2 μm. Values on graph indicate P-values of unpaired two-sample t-tests between NT and CPT-treated cells. The number of analyzed regions of interest (ROI) and replicates are denoted by n and r, respectively. *P < 0.05, **P < 0.01, and ***P < 0.001 and ****P < 0.0001. Cartoons were created in BioRender. Lahiri, S. (2025) https://biorender.com/e34f095.
