Fluorescence anisotropy DNA binding assay.

Binding assays were performed using a fluorescein-labeled 41-mer containing AAVS1 or p5 RBS sequences. The sequences used were 5′-TGGCGGCGGTTGGGGCTCGGCGCTCGCTCGCTCGCTGGGCG-3′ (AAVS1) and 5′-ACCGGGCAAAATGGAGACCCTGCGTGCTCACTCGGGCTTAA-3′ (p5), where the AAVS1 and p5 sequences are in italics (40, 41). Rep68 WT and mutant proteins at concentrations ranging from 5 nM to 3 μM were mixed with DNA (5 nM) in a final volume of 300 μl using the following buffer: 25 mM HEPES (pH 7.0), 100 mM NaCl, 1 mM TCEP. Fluorescence readings were taken on a PC1 fluorimeter (ISS, Inc.) with excitation and emission filters at 490 and 520 nm, respectively. The tubes were equilibrated at 20°C for 20 min before measurement. Each anisotropy point is the average of 10 measurements. Anisotropy is calculated as the ratio of the difference between the vertical and horizontal emission intensities to the total normalized intensity. The fraction of DNA bound (B) was calculated using the following equation: B = ([A]_x − [A]_DNA)/([A]_final – [A]_DNA), where [A]_x represents the anisotropy measured at protein concentration x, [A]_DNA is the anisotropy of free fluorescent DNA, and [A]_final is the anisotropy at saturation. The data were fit to a single binding site model by use of the Hill coefficient and the program Origin (Origin Labs). Each experiment was done in triplicate.