mCRY2 ve mBMAL1 etkileşiminin yapısal ve biyokimyasal analizi

Circadian rhythms are intrinsic periodic oscillations of physiological, biochemical and behavioral processes within approximately 24h periodicity in diverse species ranging from bacteria to mammals. CRYPTOCHROME proteins are one of the essential components of the mammalian circadian clock and null mutations of Cry leads to severe disruption of normal circadian clock function. Although forward and reverse genetic approaches have successfully unveiled the core and auxiliary circadian clock components in detail, the molecular mechanisms of the transcriptional repression in the circadian clock feedback loop remains elusive.In this thesis work, evidence will be provided through a reverse genetics approach that interaction of mCRY2 with mPER2 is mediated through evolutionary conserved amino acids on the C-terminus of mCRY2 (R501 and K503) and that interaction is essential for transcriptional repression of both endogenous and exogenous BMAL1:CLOCK heterodimer transcriptional activity. Moreover, it will be shown that mPER2 interaction is not a prerequisite for mCRY2 to bind to other circadian clock components, as ectopically expressed loss-of-function mutant immunoprecipitates with ectopically expressed mBMAL1. Furthermore, the functionality of the loss-of-function protein will be explored in terms of interaction with other core circadian clock proteins and nuclear localization pattern. It will be shown that loss-of-function is solely based on elimination of the interaction and not due to aberrant translocation of the mutant protein, as ectopically expressed tagged proteins can be detected immunocytochemically and also GFP-fusion constructs directly translocate into nucleus. In addition, the mBMAL1 interaction hot spots on mCRY2 are mapped to F171 on the N-terminus and other unresolved residues through another reverse genetics approach.