Fig. 2: Purified heterotrimeric RPA forms liquid droplets in vitro.
a, Coomassie staining of the purified trimeric human RPA complex separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). b, Spontaneous formation of spherical liquid droplets and surface wetting by the purified RPA complex. Representative stills from time-lapse microscopy are shown. For illustration purposes, one RPA droplet per image is marked by an orange dotted circle, and examples of surface wetting at the plate bottom are marked by a red dotted curved line. See Supplementary Video 2 and Methods for details. c, Example of RPA droplet fusion from time-lapse microscopy as in b. d, Stimulation of RPA condensation by ssDNA. The purified trimeric RPA complex was incubated with equimolar amounts of 40-nucleotide-oligomer ssDNA (Supplementary Table 3), or sequence-matched dsDNA, as indicated, and RPA droplet formation was analyzed. e, Turbidity measurements of purified trimeric RPA incubated with different molar ratios of 40-nucleotide-oligomer ssDNA. Turbidity measurements with n = 4 replicates were performed and normalized to control. Averages and s.d. are shown. One-way analysis of variance (ANOVA) with Dunnett’s test compared with control, 1:30, 1:10, 1:6, 1:3 ****P < 0.0001; 1:1 **P = 0.001. f, Turbidity phase diagram of RPA versus ssDNA (concentration range: 0 µM, 2.5 µM, 5 µM, 10 µM, and 15 µM for both RPA and 40-nucleotide-oligomer ssDNA). A heat map of average turbidity measurements from n = 4 replicates is shown. g, Co-assembly of ssDNA into RPA droplets. Cy3-labeled purified RPA was incubated with equimolar 17-nucleotide-oligomer FAM-labeled ssDNA. Two representative example images of RPA-ssDNA droplets are shown. h, RPA-ssDNA droplets maintain liquid properties and undergo fusion. Purified RPA was incubated with equimolar 40-nucleotide-oligomer Cy3-labeled ssDNA. Representative stills from time-lapse microscopyare shown. Scale bars, 10 µm.
