Targeting the OB-Folds of Replication Protein A with Small Molecules
Figure 3
Irreversible inhibition of RPA DBD-A/B-DNA-binding activity by CheSS19. (a) EMSA analysis of CheSS19 inhibition of RPA DBD-A/B. Assays were performed as described in the legend to Figure 2. Reactions contained 12.5 mM DNA, 50 nM DBD-A/B and 0, 25, 50, 100 and 200 μM CheSS19 (Lanes, 2-6, resp.) (b) CheSS19 inhibition of full-length heterotrimeric RPA as assessed by EMSA. Reactions were identical to those in panel (a) except contained 25 nM full-length RPA. (c) Quantification of the binding data presented in panels (a) and (b). (d) Fluorescence polarization analysis of CheSS19 inactivated RPA DBD-A/B. RPA DBD-A/B was preincubated with vehicle (1% DMSO) or CheSS19 (1 pmol in 1% DMSO) for 30 minutes at 37°C. Following incubation, the reaction mix was dialyzed versus H1 buffer overnight at 4°C. The protein was recovered, and DNA-binding activity measured by FP analysis of binding to an F-dT12 substrate was performed as described in “Section 2”. Bar 1: DNA control, Bar 2: control vehicle-treated DBD-A/B, and Bar 3: CheSS19-treated DBD-A/B. Control vehicle did not show inhibition of binding before dialysis similarly to Figure 5 Lane 3. The data represent the mean and range of two independent experiments.
