Fig. 4: The phosphorylated N-IDR of RPA2 modulates RPA phase separation properties.
a, Time-resolved optoDroplet formation of RPA2 WT, RPA2 S/T→A, and RPA2 S/T→D fused to Cry2-mCherry. Representative stills from live-cell microscopy are shown. b, Accumulated optoDroplet intensity per nucleus of WT RPA2, phosphodeficient RPA2 S/T→A, and phosphomimetic RPA2 S/T→D fused to Cry2-mCherry was analyzed and normalized to the average accumulated optoDroplet intensity of the corresponding dark condition. Two-way ANOVA with Šidák`s test, RPA2 WT, RPA2 S/T→A ****P < 0.0001; RPA2 S/T→D n.s. P = 0.9435. c, Nuclear mean intensities of Cry2-mCherry in cells analyzed in b. b,c, Averages and s.d. are shown for n = 4 independent samples (>350 cells per sample). d, Accumulated optoDroplet intensity per nucleus of WT RPA2 and phosphomimetic mutants fused to Cry2-mCherry was analyzed. Averages and s.d. are shown for n = 4 independent samples (>15 cells per sample, average 61 cells per sample). Two-way ANOVA with Šidák`s test, RPA2 WT, RPA2-S8D, RPA2-S11D, RPA2-S12D, RPA2-T21D, RPA2-S23D, RPA2-S33D, RPA2-S4D S8D, RPA2-S23D S33D, RPA2-S8D S33D, RPA2-T21D S33D, RPA2-S11D S12D S13D, RPA2-T21D S29D S33D, RPA2-S8D T21D S33D ****P < 0.0001; RPA2-T21D S23D S29D S33D ***P = 0.0003; RPA2 S/T→D n.s. P > 0.9999. e, Coomassie staining of the purified trimeric human RPA complexes (WT and S/T→D) separated by SDS–PAGE. f, Formation of liquid droplets by the purified WT RPA complex, but not by the phosphomimetic S/T→D mutant. Insets show magnifications. g, In vitro phosphorylation of the purified trimeric WT RPA complex by DNA-PK, with a western blot as a control. h, In vitro phosphorylation of the purified trimeric WT RPA complex by DNA-PK impairs RPA phase separation. Representative stills from time-lapse microscopy are shown. i, Incubation of purified trimeric Cy3-labeled RPA WT with equimolar 40-nucleotide-oligomer FAM-labeled ssDNA results in liquid droplet formation, whereas RPA S/T→D forms aggregate-like structures. Insets show magnifications. Scale bars, 10 µm.
