The yeast homeodomain protein MAT alpha 2 shows extended DNA binding specificity in complex with Mcm1

The MAT alpha 2 (alpha 2) repressor interacts with the Mcm1 protein to turn off a-cell type-specific genes in the yeast Saccharomyces cerevisiae. We compared five natural alpha 2-Mcm1 sites with an alpha 2-Mcm1 symmetric consensus site (AMSC) for their relative strength of repression and found that the AMSC functions slightly better than any of the natural sites. To further investigate the DNA binding specificity of alpha 2 in complex with Mcm1, symmetric substitutions at each position in the alpha 2 half-sites of AMSC were constructed and assayed for their effect on repression in vivo and DNA binding affinity in vitro. As expected, substitutions at positions in which there are base-specific contacts decrease the level of repression. interestingly, substitutions at other positions, in which there are no apparent base-specific contacts made by the protein in the alpha 2-DNA co-crystal structure, also significantly decrease repression. As an alternative method to examining the DNA binding specificity of alpha 2, we performed in vitro alpha 2 binding site selection experiments in the presence and absence of Mcm1. In the presence of Mcm1, the consensus sequences obtained were extended and more closely related to the natural alpha 2 sites than the consensus sequence obtained in the absence of Mcm1. These results demonstrate that in the presence of Mcm1 the sequence specificity of alpha 2 is extended to these positions.