We also generated the reverse substitution in the Dri construct, replacing the presumptive base-specific contact residues with serines. The sequence of the resultant mutant, designated Dri.SSS, as shown in Figure 5, and the DNA-binding behavior is shown in Figure 6B. Strikingly, the Dri.SSS variant maintains a clear capacity for sequence-specific binding, selecting a pattern of DNA fragments very similar to those selected by wild-type Dri. It is apparent, though, from the KCl titration in Figure 6B, that the Dri.SSS variant has reduced overall affinity for DNA. No DNA binding is detected at this exposure in the 125 mM lane, while wild-type Dri consistently shows detectable binding in similar assays to at least 200 mM KCl (Figures 3 and 6B). The most direct explanation for these results is that these positions in Dri do make significant DNA contacts that are important for affinity, but which are not major determinants of sequence specificity. The DNA-binding affinity of p270 is strong despite the presence of serines at these positions, implying that the role of individual positions is not neccesarily directly comparable between different ARIDs.
