Fig 1: The nuclease and helicase activities of DNA2 jointly safeguard cell proliferation and unrestrained helicase activity of nuclease-dead DNA2 D277A causes genotoxic stress in S and G2 phase.a, Western blot analysis (n = 1) on lysates from RPE-1 OsTIR1 DNA2dd cells upon doxycycline-induced (1 μg/ml) expression of the indicated wild-type and mutant versions of DNA2 for 8 h. KU80, loading control. b, Proliferation of RPE-1 OsTIR1 DNA2dd cells upon expression of wild type or mutant DNA2 as indicated in the presence or absence of DIA over 3 days. 1500 cells plated. Data normalised to untreated RPE-1 OsTIR1 control (Ctrl.) cells and presented as mean values ± s.d. (n = 3 independent experiments). c, Quantification of RPA foci 2, 4, and 8 h after late S phase upon treatment of the indicated cells as shown in Fig. 3a (n = 50 cells over two independent experiments at each timepoint). Note the induction of supernumerary RPA foci associated with the expression of nuclease-dead DNA2 D277A. Bars indicate the median. d, Analysis of RPA foci in RPE-1 OsTIR1 DNA2dd cells complemented or not with doxycycline-induced DNA2 versions as indicated after treatment with DIA for 16 h and pulsed with EdU (10 μM) for the final 60 min. Cell-cycle stage was determined by DAPI intensity and EdU incorporation into replicating cells. Data is presented as mean values ± s.d. (n = 3 independent experiments). e, Nuclear area measurements for the indicated numbers (n) of RPE-1 OsTIR1 control (Ctrl.) and RPE-1 OsTIR1 DNA2dd cells (representative of three independent experiments). Cells were complemented as indicated and treated with DIA for three days. Median and quartiles marked. Source data
Fig 2: Loss of DNA2 triggers ATR–p21-dependent cell-cycle exit from G2.a, Western blots (representative of three independent experiments) for DIA-treated RPE-1 OsTIR1 (Ctrl) and RPE-1 OsTIR1 DNA2dd cells. KU80, loading control. b, Nuclear area for the indicated number (n) of RPE-1 OsTIR1 (Ctrl) and RPE-1 OsTIR1 DNA2dd cells treated as indicated for 3 days. The median and quartiles are marked. ATRi, 1 μM VE-821. Two-sided Mann–Whitney U-test was applied. c, Representative images and β-galactosidase activity measurements for the indicated number (n) of RPE-1 OsTIR1 DNA2dd cells treated with DIA or palbociclib (5 μM) for the indicated times over two independent experiments. The median and quartiles are marked, and one-way analysis of variance was applied. d, Representative images and quantification of subcellular cyclin B1 localization in DIA-treated RPA-foci-positive RPE-1 OsTIR1 DNA2dd cells (n = 3 independent experiments). Error bars represent the mean ± s.d. e, Model of the essential role of DNA2. At stalled RFs, DNA2-dependent fork processing counteracts fork reversal, thereby promoting fork reactivation and faithful DNA replication. Upon the loss of DNA2, reversed forks persist and give rise to HoRReR. Excessive recombination-dependent DNA synthesis in DNA2-deprived cells results in the accumulation of ssDNA, and cells invariably respond by ATR-dependent checkpoint signalling, p21-mediated sequestration of cyclin B1 and permanent cell-cycle exit before mitosis. Scale bars, 100 μm (c), 20 μm (d).Source data
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