DNA Binding by the Male and Female Doublesex Proteins of Drosophila melanogaster*

Abstract

Drosophila yolk protein genes are regulated by doublesex male protein (DSX^M) in males and doublesex female protein (DSX^F) in females. Both proteins bind to the same DNA sites from which DSX^M represses and DSX^F activates transcription. The proteins are identical through 397 NH₂-terminal amino acids that include domains for oligomerization and DNA binding. The remaining COOH termini are sex-specific and include an essential part of a second oligomerization domain. We report here mobility shift assays that examine the DNA binding properties of purified DSX^M and DSX^F. Dimers of DSX^M and DSX^F bind to a regulatory site, dsxA, with the same affinity (K_app = 0.2 nM), specificity (specific/nonspecific ≈ 1.2 × 10⁴), and dependence on monovalent and divalent cations. The DNA association rate constants also are indistinguishable (k_on = 4.6 × 10⁶ M⁻¹ s⁻¹) as are the several terms of the dissociation reaction. Dissociation has an intrinsic rate of k_off = 5.1 × 10⁻⁴ s⁻¹ and other rate terms that depend on the free concentration of specific DNA binding sites (2.4 × 10⁴ M⁻¹ s⁻¹) or nonspecific binding sites (2.4 M⁻¹ s⁻¹). This first order dependence on unbound DNA suggests that a direct transfer between DNAs is likely to occur when DSX proteins search for specific sites in the many short open DNA regions of chromatin. Overall, dimer binding to individual DNA sites appears to be determined by the sex-nonspecific part of the two proteins. We infer that the sex-specific oligomerization domains play roles in binding cooperativity to multiple DNA sites or in other protein:protein interactions.