Mutational analysis suggests that circadian period-altering mutations of DBT affect Interactions of DBT with other circadian Proteins. Anandakrishnan Venkatesan¹, Michael Muskus², Ed Bjes¹, Jeffrey Price¹. 1) University of Missouri Kansas City, Kansas City, MO; 2) Washington University, St Louis, MO.

   Mutational analysis of DBT is addressing if its effects on circadian period are determined in part by interactions with other proteins. The period-altering mutations of DBT possess lower kinase activity but have different effects on period. We hypothesized that if these mutations alter period by affecting interaction of DBT with the other circadian partners then these effects should persist in a kinase-inactive background (DBT^K/R - dominant negative). To test this hypothesis we made double mutants combining both dbt^K/R and each of the period altering mutants in cis. All three double mutants reduce the long period of the dbt^K/R;dbt^WT genotype and partially restore oscillations of PER phosphorylation. The period-shortening of the by these mutations suggests that they affect the period by a mechanism in part independent of kinase activity (e.g. interaction with other clock components). The TAU mutation producing a short period (21 hrs) has been proposed to be party of a triad that binds phospho-amino acids, thereby targeting DBT to sites that have already been phosphorylated. We hypothesized that the amino acids around the TAU mutation might also be involved in this process or in producing other protein-protein interactions. We made mutations in amino acids that were closer to this triad as well as those and that were away from it. The mutations that were close to the triad produced a short period (~16 - 22 hrs) and the mutations that were farther away produced a longer period. The shortening of period by some mutations suggests that they affect a TAU-like domain involved in a protein-protein interaction that lengthens period. The identification of the interacting partners is underway.