Fluorescence Anisotropy DNA Binding Assay.

Binding assays were performed using 5 nM fluorescein-labeled 26-mer Ter3 DNA. Reb1 constructs at different concentrations were mixed with DNA at a final volume of 300 µL using the following buffer: 25 mM Hepes, pH 7.0, 100 mM NaCl, and 1 mM TCEP. Fluorescence readings were taken on a PC1 fluorimeter (ISS) with excitation and emission filters at 490 and 520 nm. Tubes were equilibrated at 20 °C for 20 min before measurement. Each anisotropy point is the average of 10 measurements. Anisotropy was calculated as the ratio of the difference between vertical and horizontal emission intensities over the total normalized intensity. The fraction of DNA bound (B) was calculated using

where [A]_x represents the anisotropy measured at protein concentration X, [A]_DNA is the anisotropy of free fluorescence DNA, and [A]_FINAL is the anisotropy at saturation. Data were fit to a single binding site model using the program Origin (Origin Labs). Each experiment was done in triplicate. Titrations for MybADs and Myb repeats proteins were carried out to a concentration of 3 µM to reach saturation.