Fig. 1
From: Replication fork reversal triggers fork degradation in BRCA2-defective cells
Stalled replication forks can reverse in the absence of BRCA2, but are targeted by nucleolytic degradation. a, c Electron micrographs of representative replication forks from RPE-1 cells: parental (P) and daughter (D) duplexes. a The black arrow indicates the regressed arm (R); the four-way junction at the reversed fork is magnified in the inset. c The white arrow points to a ssDNA region at the fork. Scale bar, 200 nm ( = 460 bp), 40 nm ( = 92 bp) in the inset. b Left panel: frequency of reversed replication forks isolated from mock-depleted (siLuc) and BRCA2-depleted (siBRCA2) RPE-1 cells upon optional 5 h treatment with 4 mM HU; where indicated, 50 μM mirin was added 1 h before HU treatment (6 h total treatment). The number of replication intermediates analyzed is indicated in parentheses. The graph depicts mean and SD from three independent EM experiments, blinded to the investigator. The results of the individual biological replicates are in Supplementary Table 1. Right panel: western blot analysis of BRCA2 levels in siLuc and siBRCA2 RPE-1 cells, 48 h after transfection. TFIIH, loading control. d Graphical distribution of ssDNA length at the junction (white arrow in Fig. 1c) in siLuc and siBRCA2 RPE-1 cells optionally treated with 4 mM of HU for 5 h and 50 μM of mirin for 6 h. Only molecules with detectable ssDNA stretches are included in the analysis. The lines show the median length of ssDNA regions at the fork in the specific set of analyzed molecules. Statistical analysis: Mann–Whitney test; ns, not significant; *P < 0.1; **P < 0.01; ***P < 0.001; ****P < 0.0001. The number of analyzed molecules is in brackets
