17 research outputs found
Mammalian TIMELESS Is Involved in Period Determination and DNA Damage-Dependent Phase Advancing of the Circadian Clock
Mammalian TIMELESS Is Involved in Period Determination and DNA Damage-Dependent Phase Advancing of the Circadian Clock
The transcription/translation feedback loop-based molecular oscillator underlying the generation of circadian gene expression is preserved in almost all organisms. Interestingly, the animal circadian clock proteins CRYPTOCHROME (CRY), PERIOD (PER) and TIMELESS (TIM) are strongly conserved at the amino acid level through evolution. Within this evolutionary frame, TIM represents a fascinating puzzle. While Drosophila contains two paralogs, dTIM and dTIM2, acting in clock/photoreception and chromosome integrity/photoreception respectively, mammals contain only one TIM homolog. Whereas TIM has been shown to regulate replication termination and cell cycle progression, its functional link to the circadian clock is under debate. Here we show that RNAi-mediated knockdown of TIM in NIH3T3 and U2OS cells shortens the period by 1 hour and diminishes DNA damage-dependent phase advancing. Furthermore, we reveal that the N-terminus of TIM is sufficient for interaction with CRY1 and CHK1 as well for homodimerization, and the C-terminus is necessary for nuclear localization. Interestingly
Dimerization and nuclear entry of mPER proteins in mammalian cells
Nuclear entry of circadian oscillatory gene products is a key step for the
generation of a 24-hr cycle of the biological clock. We have examined
nuclear import of clock proteins of the mammalian period gene family and
the effect of serum shock, which induces a synchronous clock in cultured
cells. Previously, mCRY1 and mCRY2 have been found to complex with PER
proteins leading to nuclear import. Here we report that nuclear
translocation of mPER1 and mPER2 (1) involves physical interactions with
mPER3, (2) is accelerated by serum treatment, and (3) still occurs in
mCry1/mCry2 double-deficient cells lacking a functional biological clock.
Moreover, nuclear localization of endogenous mPER1 was observed in
cultured mCry1/mCry2 double-deficient cells as well as in the liver and
the suprachiasmatic nuclei (SCN) of mCry1/mCry2 double-mutant mice. This
indicates that nuclear translocation of at least mPER1 also can occur
under physiological conditions (i.e., in the intact mouse) in the absence
of any CRY protein. The mPER3 amino acid sequence predicts the presence of
a cytoplasmic localization domain (CLD) and a nuclear localization signal
(NLS). Deletion analysis suggests that the interplay of the CLD and NLS
proposed to regulate nuclear entry of PER in Drosophila is conserved in
mammals, but with the novel twist that mPER3 can act as the dimerizing
partner
The Potorous CPD Photolyase Rescues a Cryptochrome-Deficient Mammalian Circadian Clock
Despite the sequence and structural conservation between cryptochromes and photolyases, members of the cryptochrome/photolyase (flavo)protein family, their functions are divergent. Whereas photolyases are DNA repair enzymes that use visible light to lesion-specifically remove UV-induced DNA damage, cryptochromes act as photoreceptors and circadian clock proteins. To address the functional diversity of cryptochromes and photolyases, we investigated the effect of ectopically expressed Arabidopsis thaliana (6-4)PP photolyase and Potorous tridactylus CPD-photolyase (close and distant relatives of mammalian cryptochromes, respectively), on the performance of the mammalian cryptochromes in the mammalian circadian clock. Using photolyase transgenic mice, we show that Potorous CPD-photolyase affects the clock by shortening the period of behavioral rhythms. Furthermore, constitutively expressed CPD-photolyase is shown to reduce the amplitude of circadian oscillations in cultured cells and to inhibit CLOCK/BMAL1 driven transcription by interacting with CLOCK. Importantly, we show that Potorous CPD-photolyase can restore the molecular oscillator in the liver of (clock-deficient) Cry1/Cry2 double knockout mice. These data demonstrate that a photolyase can act as a true cryptochrome. These findings shed new light on the importance of the core structure of mammalian cryptochromes in relation to its function in the circadian clock and contribute to our further understanding of the evolution of the cryptochrome/photolyase protein family