Fig. 6: Formation of RAD51 filaments on CST-coated ssDNA.

a Electron microscopy with negative staining was used to observe the formation of RAD51 filaments on CST- or RPA-bound ssDNA under the condition of 150 mM KCl. The 80-nt ssDNA substrate was incubated with CST (i), RPA (ii) or, as a control, RAD51 (iii). RAD51 was then added to CST-bound ssDNA (iv) or RPA-bound ssDNA (v). Representative images from panels (i) to (v) are shown. N = 2 biologically independent experiments. b RAD51 filament length was measured in Image J software. The quantitative graph was generated in GraphPad Prism. Boxes represent the 25th and 75th percentiles and encompass the median, with whiskers showing the minimum and maximum values. N values represent the number of RAD51 filaments identified in numerous electron microscopy images collected from two independent experiments. NS not significant, ****P < 0.0001 as calculated by one-way ANOVA with Kruskal–Wallis test followed by Dunn’s post hoc test. The median values for RAD51, CST→RAD51, RPA→RAD51 are 96.41, 99.17, and 67.83 nm, respectively. Note that some filaments are much longer than expected due to end-to-end association of two filaments⁶⁹. c RAD51 assembly on CST- or RPA-bound ssDNA was monitored by smFRET under the condition of 150 mM KCl. A 35-nt overhang DNA substrate was used for smFRET, as shown in Fig. 2a. The smFRET states of RAD51-, RPA-, and CST-ssDNA are ~0.05 (i), ~0.15 (ii), and ~0.2 (iv), respectively. Although addition of RAD51 did not alter the smFRET state of RPA-bound ssDNA (~0.15, iii), the smFRET state shifted from 0.2 to 0.05 for CST-bound ssDNA (v). This outcome indicates that RAD51 can access CST-bound ssDNA. N values represent the number of individual molecules collected from at least three independent experiments and are displayed in the upper right corner. d Representative time-course of RAD51 assembly on CST-coated ssDNA. RAD51 was introduced to the preformed surface-bound CST-ssDNA (gray). Before transitioning into the RAD51-bound smFRET state (pink), a high-FRET intermediate state (green) was observed. Rastergram of 126 molecules showing a similar pattern. e Kinetics analysis showing that RAD51 assembled on CST-coated ssDNA in 64 ± 2.2 s (N = 96), whereas it was predicted to take thousands of minutes to do so on RPA-coated ssDNA (N = 13). Nearly no RAD51 assembly was observed for RPA-coated ssDNA. Data are plotted as a best-fit result ± 95% C.I.