FIGURE 9.
Sequence-specific DNA binding. A, fluorescence donor emission of 6-FAM/TAMRA-labeled DNA site (black line) was attenuated due to increased FRET efficiency on binding to WT SRY domain (filled circles). Donor emission was only slightly affected on addition of an unrelated DNA duplex (derived from λ operator site OL1; open triangles) in 10-fold excess, indicating negligible competition. B, emission spectra of specific FRET-labeled WT protein·DNA complex (filled circles) exhibited ∼50% reduction in donor emission due to partial dissociation in the presence of an equimolar specific unlabeled DNA site (open triangles). C and E, similar trends were observed for variant complexes W70A (gray, C) and W70L (red, E) on addition of the λ DNA site in 10-fold excess. D and F, enhanced donor emission on partial dissociation of FRET-labeled W70A and W70L domain·DNA complexes (gray and red, respectively) on addition of equimolar-specific unlabeled DNA site. arb. units, arbitrary units.