The yeast a1 and alpha 2 homeodomain proteins do not contribute equally toheterodimeric DNA binding

In diploid cells of the yeast Saccharomyces cerevisiae, the alpha 2 and al homeodomain proteins bind cooperatively to sites in the promoters of haploi d cell-type-specific genes (hsg) to repress their expression. Although both proteins bind to the DNA, in the alpha 2 homeodomain substitutions of resi dues that are involved in contacting the DNA have little or no effect on re pression in vivo or cooperative DNA binding with al protein in vitro. This result brings up the question of the contribution of each protein in the he terodimer complex to the DNA-binding affinity and specificity. To determine the requirements for the a1-alpha 2 homeadomain DNA recognition, we system atically introduced single base-pair substitutions in an a1-alpha 2 DNA-bin ding site and examined their effects on repression in vive and DNA binding in vitro. Our results show that nearly all substitutions that significantly decrease repression and DNA-binding affinity are at positions which are sp ecifically contacted by either the alpha 2 or a1 protein. Interestingly, an alpha 2 mutant lacking side chains that make base-specific contacts in the major groove is able to discriminate between the wild-type and mutant DNA sites with the same sequence specificity as the wild-type protein. These re sults suggest that the specificity of alpha 2 DNA binding in complex,vith a 1 does not rely solely an the residues that make base-specific contacts. We have also examined the contribution of the a1 homeodomain to the binding a ffinity and specificity of the complex. In contrast to the lack of a defect ive phenotype produced by mutations in the alpha 2 homeodomain, many of the alanine substitutions of residues in the a1 homeodomain have large effects on a1-alpha 2-mediated repression and DNA binding. This result shows that the two proteins do not make equal contributions to the DNA-binding affinit y of the complex.