DNA binding by the male and female doublesex proteins of Drosophila melanogaster.
Drosophila yolk protein genes are regulated by doublesex male protein (DSXM) in males and doublesex female protein (DSXF) in females. Both proteins bind to the same DNA sites from which DSXM represses and DSXF 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 DSXM and DSXF. Dimers of DSXM and DSXF bind to a regulatory site, dsxA, with the same affinity (Kapp = 0.2 nM), specificity (specific/nonspecific approximately 1.2 x 10(4)), and dependence on monovalent and divalent cations. The DNA association rate constants also are indistinguishable (kon = 4.6 x 10(6) M-1 s-1) as are the several terms of the dissociation reaction. Dissociation has an intrinsic rate of koff = 5.1 x 10(-4) s-1 and other rate terms that depend on the free concentration of specific DNA binding sites (2.4 x 10(4) 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.
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Rosenstiel Center and the Graduate Department of Biochemistry, Brandeis University, Waltham, Massachusetts 02254-9110, USA.
Wensink
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D000818:Animals; D001667:Binding, Competitive; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D029721:Drosophila Proteins; D004331:Drosophila melanogaster; D005260:Female; D007301:Insect Hormones / Q000378:metabolism*; D007700:Kinetics; D008297:Male; D012965:Sodium Chloride
D004268:DNA-Binding Proteins; C070369:DSX protein, Drosophila; D029721:Drosophila Proteins; D007301:Insect Hormones; D012965:Sodium Chloride; D004247:DNA