Figure 3.

Off-rate analysis for E2c–E2-DBS complex. The binding of E2c to fluorescein-labeled F-E2-DBS (10 nM double stranded) was measured as a function of the increasing in fluorescence polarization. After ≈10 min of equilibration, 10 nM E2c was added to DNA. After ≈10 min of incubation, the dissociation process was analyzed as a function of time on a continuous measurement of fluorescence polarization, following the addition of a 100-fold excess (for a final concentration of 1 μM double-stranded DNA) of unlabeled competitor: (A) E2-DBS; (B) poly(A-T) 18-mer. Experiments were carried out in 50 mM Bis-Tris⋅HCl/1 mM DTT/200 mM NaCl (pH 7.0) at 22°C. In all cases, maximal dilution was 1.5%.