LINKAGE BETWEEN OLIGOMERIZATION AND DNA-BINDING IN DROSOPHILA DOUBLESEX PROTEINS

The doublesex gene of Drosophila melanogaster encodes DSXM protein in males and DSXF protein in females. Dimers of each protein bind a DNA s ite from which DSXM represses and DSXF activates transcription. Amino acids 1-397 are identical between the proteins and include a domain (D BD) for both DNA binding and protein oligomerization. The remaining no nhomologous and therefore sex-specific C-termini include an essential part of a second oligomerization domain. We have used mobility shift a ssays to investigate the effects these three oligomerization domains ( DBD and two sex-specific) have on DSX dimerization and DNA binding. Th e intrinsic DNA binding affinities of DSXM and DSXF dimers are indisti nguishable from each other (0.17 +/- 0.04 nM) and slightly lower than that of DBD dimers (0.48 nM). In contrast, the dimerization dissociati on constants of DSXM (0.05 +/- 0.02 nM) and DSXF (0.16 +/- 0.05 nM) ar e slightly different, but 4 orders of magnitude lower than that of DBD (430 nM). Thus sequences outside of DBD, presumably the sex-specific oligomerization domains, have substantial effects on apparent DNA bind ing affinity through thermodynamically linked effects on dimerization of full-length proteins. Further, when two DNA binding sites are adjac ent, DBD dimers show no binding cooperativity, whereas full-length dim ers bind with 2-fold different cooperativity (DSXF, k(12) = 2.6; DSXM k(12) = 5.4). This suggests that the sex-specific domains may have a s econd effect on DNA binding, namely, an effect on binding cooperativit y that depends on the number and arrangement of DNA sites.