Date of Award
Doctor of Philosophy
Andrew C Liu
Circadian clocks in mammals are based on a negative feedback loop in which transcriptional repression by the Cryptochromes, CRY1 and CRY2, lies at the heart of the mechanism. Despite similarities in their sequence, domain structure and biochemical activity, they play distinct roles in the mammalian clock function. However, detailed biochemical studies have not been straightforward and function of Cryptochrome (Cry) has not been examined in real clock cells using kinetic measurements. In this study, we demonstrate, through cell-based genetic complementation and real-time molecular recording, that Cry1 alone is able to maintain cell-autonomous circadian rhythms, while Cry2 cannot. Using this novel functional assay, we identify a Cryptochrome differentiating α-helical domain within the photolyase homology region (PHR) of CRY1 protein, designated as CRY1-PHR(313-426), that is required for clock function and distinguishes CRY1 from CRY2. Further, in contrast to speculation, we demonstrate that the divergent carboxyl-terminal tail domain (CTD) is dispensable for circadian clock function, but it serves to modulate rhythm amplitude and period length. Finally, we identify the biochemical basis of their distinct function; CRY1 is a much more potent repressor of BMAL1/CLOCK transcriptional activity than CRY2, and the strength of repression by various forms of CRY proteins significantly correlates with rhythm amplitude. Taken together, our results demonstrate that the CRY1-PHR(313-426), not the divergent CTD, is critical for clock function. These findings provide novel insights into the evolution of the diverse functions of the photolyase/cryptochrome family of flavoproteins and offer new opportunities for mechanistic studies of CRY function.
dissertation or thesis originally submitted to the local University of Memphis Electronic Theses & dissertation (ETD) Repository.
Khan, Sanjoy Kumar, "Genetic and Biochemical Dissection of Differential Functions of Cryptochrome 1 and 2 in the Mammalian Circadian Clock" (2012). Electronic Theses and Dissertations. 603.