Supplementary Figure 9: The importance of the physical interactions between RPA and Rad52 as well as Rad52 and ssDNA for the modulation of RPA domain dynamics.

The activities of the RAD52 on the RPA-coated ssDNA depend on the species-specific interaction between the two proteins ¹¹. To determine whether the physical interaction between Rad52 and RPA plays a role in the Rad52-mediated change in the RPA conformational dynamics, we carried out the single-molecule analysis of the yeast RPA dynamics in the presence of human RAD52. (a) Experimental scheme for visualization of the effect of hRAD52 on RPA-DBD-D dynamics. Binding of the fluorescently-labeled RPA (100pM) to the ssDNA (blue line) brings the MB543 fluorophore within the evanescent field and its excitation. NA – neutravidin, b – biotin. (b) Human RAD52 protein shares the conserved N-terminal domain with the yeast protein. This domain is responsible for the formation of the oligomeric ring and for the interaction with DNA. Their C-terminal, protein-protein interaction domains are highly different resulting in the absence of cross-species interactions with RPA. (c) Representative fluorescence trajectories depicting the conformational dynamics of the individual RPA molecules labeled within RPA-DBD-D. After replacement of RPA in the reaction chamber with 700pM hRAD52, the same four conformational states are observed in RPA-DBD-D^MB543 trajectories suggesting that human RAD52 does not affect the yeast RPA conformational dynamics. Therefore, we conclude that the Rad52-RPA physical interaction is required for the observed effect. (d). Electrophoretic mobility shift assay (EMSA) of Cy5-labeled ssDNA (30nt) contrasting increasing concentrations of scRad52 vs hRAD52 added to scRPA coated ssDNA. Table includes reaction conditions for each lane. Reactions in the bottom portion were crosslinked using 0.1% glutaraldehyde. The species of each band are identified on the right of the gel. The results of this EMSA experiment suggest that the interaction between hRAD52 and scRPA-coated ssDNA are similar to that between scRad52 and the scRPA-coated ssDNA. Thus, the absence of the DBD-D modulation in the single-molecule control experiment is solemnly due to the absence of the protein-protein interaction between the ssDNA-bound scRPA and hRAD52. Previously, we identified epigallocatechin (EGC) as a specific inhibitor of the human RAD52 interaction with ssDNA ¹². In contract, EGC displays no activity towards human RPA ¹². Here, we confirmed that EGC also inhibits S. cerevisiae Rad52-ssDNA interaction and has no effect on the interaction between S. cerevisiae RPA and ssDNA. We therefore, used EGC to determine whether the interaction between Rad52 and ssDNA is important for the modulation of the RPA conformational dynamics. (e) FRET-based inhibitor titration of EGC into solution of 100 nM Rad52 and 10 nM Cy3-dT30-Cy5 ssDNA. FRET begins high where the FRET labeled DNA is wrapped around Rad52, bringing Cy3 and Cy5 into proximity and decreases as inhibitor prevents Rad52 ssDNA binding. The IC50 value calculated for inhibition of Rad52 ssDNA binding is shown below the curve. (f) FRET-based inhibitor titration of EGC into solution of 10 nM RPA and 10 nM Cy3-dT30-Cy5 ssDNA. The absence of FRET increase with EGC titration indicates that EGC does not inhibit RPA ssDNA binding. (g) Scheme depicting interactions between RPA and Rad52 and DNA. Addition of ECG inhibits Rad52 ssDNA binding. (h) Epigallocatechin (ECG), an inhibitor of Rad52 ssDNA binding. (i) Experimental scheme for visualization of the effect of Rad52 with 10uM EGC on RPA-DBD-D dynamics. Binding of the fluorescently-labeled RPA (100 pM) to the ssDNA (blue line) brings the MB543 fluorophore within the evanescent field and its excitation. NA – neutravidin, b – biotin. (j) Representative fluorescence trajectories depicting the conformational dynamics of the individual RPA molecules labeled within RPA-DBD-D. After replacement of RPA in the reaction chamber with 700 pM Rad52 and 10 uM EGC, the same four conformational states are observed in RPA-DBD-D^MB543 trajectories. (k) Comparison of the lifetimes of the individual states and fractional visitation to each state available to RPA-DBD-D^MB543 alone (grey) and in the presence of Rad52 (blue) and in the presence of Rad52 and EGC (red). Data from a single experiment was separated into three portions and each portion was analyzed and plotted separately. The presence of EGC returned almost all the lifetimes and all the visitation frequencies to the same values as were obtained in the absence of Rad52. Most importantly, we observed the reemergence of the state 4, suggesting that the Rad52-ssDNA interaction is important for the formation of the RPA-ssDNA-Rad52 complex that modulates the accessibility of the 3′ ssDNA region occluded by RPA. A slight increase in the lifetime of the least fluorescent state 1 is likely the result of the interaction between Rad52 and RPA in the absence of the Rad52-ssDNA interaction.

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