Fig. 1: GRB2 at the DNA replication fork inhibits MRE11 mediated fork-degradation. | Nature Communications

Fig. 1: GRB2 at the DNA replication fork inhibits MRE11 mediated fork-degradation.

From: GRB2 stabilizes RAD51 at reversed replication forks suppressing genomic instability and innate immunity against cancer

Fig. 1

a FLIM/FRET showing GRB2-PCNA direct interaction within an apparent PCNA replication focus. Colocalization shown in the left, while false colored lifetime image on the right generated by pixel-by-pixel mapping of the measured lifetime-values represented by the scale 1.7–2.2 nanoseconds. Scale bar 25 μm (b) iPOND assay showing GRB2 association with replication DNA, and enriched on the nascent DNA in response replication stress. PCNA was used as a positive control. c Enhanced fork degradation in GRB2-depleted HeLa cells under replication stress. d An independent set of experiments with GRB2 reconstitution. Only WTGRB2 (KO + GRB2), but not the K109R mutant alleviated replication stress induced fork degradation in GRB2-depleted HeLa cells. Radiometric analysis of ≥200 fibers, n = 3. e Replication stress induced increased ssDNA in GRB2-depleted cells under replication stress indicating enhanced nuclease activity in HeLa cells. Scale bar 10 μm. f Quantitation of the ssDNA intensities from three independent experiments represented in (e), intensities of 100–300 foci, n = 3. g Fork degradation is inhibited by MRE11 nuclease inhibitor Mirin in HeLa cells lacking GRB2 and under replication stress. h MRE11knockddown prevents fork degradation in GRB2-deplered HeLa cells under replication stress. Fiber assay radiometric analysis of ≥ 200 fibers, n = 3 in (c, d, f–h). The significance was analyzed by two-sided Student’s t test. ***P ≤ 0.001, and ****P ≤ 0.0001; NS not significant. Error bars showing standard deviations (SD). For (b–d) and (f–h), source data are provided as a Source Data file.

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