Project 4: Structure/activity analysis of mammalian clock components by saturation mutagenesis

Dr. Carla Green and Dr. John Hogenesch, Project Leaders

Although many components of the central clock mechanism have been identified, very little is known about the biochemical details of their action. In order to further refine the molecular oscillator model and to better understand how the circadian clock controls rhythmicity, we propose to perform saturation mutagenesis to generate allelic series of the central clock components and to analyze their functions in biochemical and cell-based assays.

Random mutagenesis will be used to create libraries of random mutations in Bmal1, Clock, Cryptochrome 1, Cryptochrome 2, Period 1, and Period 2. The mutant proteins will be analyzed in high-throughput cell-based assays for alterations in their activity. The mutants of interest will be analyzed further, using a host of biochemical and cell assays to study protein-protein interactions, DNA-binding, post-translational modifications and intracellular localization. Information about the nature of the mutations and their corresponding activities will be used to identify structural features or domains that are important for specific aspects of their functions. The mutants of interest will be analyzed for effects on circadian properties (period, amplitude of rhythmicity, etc) in cycling cell assays containing per2::luciferase reporters. The definition of these functional domains will aid in the biochemical analysis of the response to small molecule perturbations and will allow better interpretations of the functions of the human genetic variants identified. Finally, these mutant forms could provide genetic tools for future manipulation of the mammalian circadian system.