We report here our systematic studies of the dynamics of four redox states of the flavin cofactor in both photolyases and insect Type 1 cryptochromes. With femtosecond resolution, we observed ultrafast photoreduction of oxidized state (FAD) in subpicosecond and of neutral radical semiquinone (FADH • ) in tens of picoseconds through intraprotein electron transfer mainly with a neighboring conserved tryptophan triad. Such ultrafast dynamics make these forms of flavin unlikely to be the functional states of the photolyase/cryptochrome family. In contrast, we find that upon excitation the anionic semiquinone (FAD •-) and hydroquinone (FADH -) have longer lifetimes that are compatible with high-efficiency intermolecular electron transfer reactions. In photolyases, the excited active state (FADH -* ) has a long (nanosecond) lifetime optimal for DNA-repair function. In insect Type 1 cryptochromes known to be blue-light photoreceptors the excited active form (FAD •-* ) has complex deactivation dynamics on the time scale from a few to hundreds of picoseconds, which is believed to occur through conical intersection(s) with a flexible bending motion to modulate the functional channel. These unique properties of anionic flavins suggest a universal mechanism of electron transfer for the initial functional steps of the photolyase/cryptochrome blue-light photoreceptor family.

a b s t r a c tMammalian cells possess a cell-autonomous molecular clock which controls the timing of many biochemical reactions and hence the cellular response to environmental stimuli including genotoxic stress. The clock consists of an autoregulatory transcription-translation feedback loop made up of four genes/proteins, BMal1, Clock, Cryptochrome, and Period. The circadian clock has an intrinsic period of about 24 h, and it dictates the rates of many biochemical reactions as a function of the time of the day. Recently, it has become apparent that the circadian clock plays an important role in determining the strengths of cellular responses to DNA damage including repair, checkpoints, and apoptosis. These new insights are expected to guide development of novel mechanism-based chemotherapeutic regimens.

It is commonly thought that disruption of the circadian clock increases the cancer incidence in humans and mice. However, it was found that disruption of the clock by the Cryptochrome (Cry) mutation in mice did not increase cancer rate in the mutant mice even after exposing the animals to ionizing radiation. Therefore, in this study we tested the effect of the Cry mutation on carcinogenesis in a mouse strain prone to cancer because of a p53 mutation, with the expectation that clock disruption in this sensitized background would further increase cancer risk. Paradoxically, we find that the Cry mutation protects p53 mutant mice from the early onset of cancer and extends their median lifespan Ϸ50%, in part by sensitizing p53 mutant cells to apoptosis in response to genotoxic stress. These results suggest alternative therapeutic approaches in management of cancers associated with a p53 mutation.apoptosis ͉ circadian clock ͉ DNA repair

Background: -catenin mutations that constitutively activate the canonical Wnt signaling have been observed in a subset of hepatocellular carcinomas (HCCs). These mutations are associated with chromosomal stability, low histological grade, low tumor invasion and better patient survival. We hypothesized that canonical Wnt signaling is selectively activated in well-differentiated, but repressed in poorly differentiated HCCs. To this aim, we characterized differentiation status of HCC cell lines and compared their expression status of Wnt pathway genes, and explored their activity of canonical Wnt signaling.

Cryptochrome (CRY) is a blue-light sensitive flavoprotein that functions as the primary circadian photoreceptor in Drosophila melanogaster. The mechanism by which it transmits the light signal to the core clock circuitry is not known. We conducted in vitro studies on the light-induced conformational change in CRY and its effect on protein-protein interaction and performed in vivo analysis of the lifetime of the signaling state of the protein to gain some insight into the mechanism of phototransduction. We find that exposure of CRY to blue light induces a conformation similar to that of the constitutively active CRY mutant with a C-terminal deletion (CRYΔ). This light-induced conformation has a half-life of ∼15 min in the dark at 25°C and is characterized by increased affinity to Jetlag E3 ligase. In vivo analysis reveals that in the Drosophila S2 cell line, the signaling state induced by a millisecond light exposure has a half-life of 27 min in the dark at 0°C during which period it is susceptible to degradation by the ubiquitin-proteasome system. These findings lead to a plausible model for circadian photoreception/ phototransduction in Drosophila.circadian clock | photocycle | proteolysis | sensory flavoprotein C ryptochrome (CRY) is a flavoprotein that regulates growth and development in plants in response to blue light, functions as a circadian photoreceptor in Drosophila and other insects, and acts as a core component of the molecular clock in mammalian organisms (1-4). Despite extensive research on CRYs photosensory function in Arabidopsis and Drosophila, its mechanism of photoreception/phototransduction is poorly understood. Even the redox status of the FAD cofactor is a matter of some debate (5-8). In the phylogenetically related protein, DNA photolyase, photoinduced cyclic electron transfer from the FADH − cofactor to a pyrimidine photodimer repairs the DNA damage and regenerates the FADH − for new rounds of catalysis (9-11). However, there is no evidence so far for a similar reaction in either Arabidopsis CRY1 (AtCRY1) and CRY2 or Drosophila CRY, which at present are the most extensively studied CRYs. The lack of evidence for a cyclic redox reaction in CRYs has led to consideration of the mechanisms of other photosensory flavoproteins as potential models for CRYs in general and Drosophila CRY in particular.Currently, three types of photosensory flavoproteins are known (12): the photolyase/CRY family, the LOV domain proteins such as phototropin, and the BLUF domain proteins such as the photoactivated adenylyl cyclase. Whereas photolyase, as noted above, carries out catalysis by light-induced cyclic electron transfer, LOV and BLUF domain proteins initiate photosignaling by a lightinduced conformational change. Failing to obtain any evidence for CRY signaling by a photolyase-like mechanism, we considered the possibility that CRY also may carry out its light signaling by a light-induced conformational change that would affect the interaction of CRY with downstream signal transduction partners (13,14). Inde...