Extended Data Fig. 4: Construction and complementation of DNA2^dd cells.

a, Schematic of the double-degron construct knock-in at endogenous DNA2 with diagnostic PCR (representative of two independent experiments) on genomic DNA from RPE-1 OsTIR1 (Ctrl.) cells and RPE-1 OsTIR1 DNA2^dd cells confirming correct integration. PCR3 amplifies an unrelated locus. Right arm and Left arm indicate the homologous regions used for gene targeting. b, Complementation of proliferation defects caused by DNA2^dd degradation. Sleeping Beauty plasmids (pSB) allowing doxycycline (Dox)-inducible expression of either luciferase or DNA2 (as shown by a western blot representative of three independent experiments) were integrated into RPE-1 OsTIR1 DNA2^dd cells. 500 cells were plated and grown in the presence of Dox (1 μg/ml) and Asv (3 μM) for 6 days prior to fixation, staining, and counting nuclei. Quantification of the results shows the mean ± s.d. of n = 3 independent experiments. Data normalised to untreated conditions. Statistical analysis by two-sided t-test with Welch’s correction. c, Complementation of colony forming defects caused by DNA2^dd degradation. Cell lines and drug treatment as in panel b. Representative image and quantification showing mean ± s.d. of n = 3 independent experiments. d, Flow cytometric analysis of RPE-1 OsTIR1 DNA2^dd following 60 h treatment ± ATRi (VE-821; 1 μM) ± DIA (n = 1). e, Cell viability of RPE-1 OsTIR1 DNA2^dd cells treated as in panel d, as measured by trypan blue exclusion. Treatment with etoposide (50 μM) for 60 h to induce cell death serves as control. Quantification of the results shows mean ± s.d. of n = 3 independent experiments.

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