Fig. 3: RPA2 is the main driver of RPA clustering.
a, Time-resolved optoDroplet formation of RPA1, RPA2, and RPA3 fused to Cry2-mCherry. Representative stills from live-cell microscopy are shown. Scale bars, 10 µm. b, Accumulated optoDroplet intensity per nucleus of single RPA subunits RPA1, RPA2, or RPA3 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, RPA3 ****P < 0.0001; RPA1 not significant (n.s.) P = 0.9998. 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 (cell number: RPA1dark n1 = 143, n2 = 168, n3 = 175, n4 = 114; RPA1light n1 = 203, n2 = 200, n3 = 241, n4 = 225; RPA2dark n1 = 937, n2 = 943, n3 = 855, n4 = 768; RPA2light n1 = 1,092, n2 = 947, n3 = 1,137, n4 = 1,024; RPA3dark n1 = 1,093, n2 = 1,231, n3 = 1,005, n4 = 814; RPA3light n1 = 1,199, n2 = 1,447, n3 = 1,643, n4 = 1,424). A.U., arbitrary units. d, GFP co-IP from naive U-2 OS cells and from U-2 OS cells stably expressing GFP-RPA2 to probe for specific RPA2 self-interaction. Endogenous RPA2 and GFP-RPA2 were detected with an anti-RPA2 antibody. e, AlphaFold66 protein structure prediction for RPA2. Consistent with X-ray crystallography data on the RPA trimerization core, two globular domains, a flexible linker, and an N-IDR were predicted. Putative and confirmed phosphorylation sites within the N-IDR are indicated. f, AlphaFold Predicted Aligned Error (PAE) heatmap depicting the distance error for every pair of residues as an estimate of position error for the structure prediction shown in e. RPA2 residue numbers run along the vertical and horizontal axes, and the color indicates PAE values for the corresponding pair of residues. g, RPA2 protein disorder prediction by GlobPlot 2.3, an online tool plotting the tendency within query proteins for order/globularity and disorder30. Disorder propensity is shown on the y axis, and amino acid position of RPA2 is on the x axis. h, RPA2 protein disorder prediction by PONDR (Predictor of Natural Disordered Regions)31. PONDR score is shown on the y axis, and amino acid position of RPA2 is on the x axis. Highly disordered regions are marked by the black bar.
