The photolyase-blue-light photoreceptor family is composed of cyclobutane pyrimidine dimer (CPD) photolyases, (6-4) photolyases, and blue-light photoreceptors. CPD photolyase and (6-4) photolyase are involved in photoreactivation for CPD and (6-4) photoproducts, respectively. CPD photolyase is classified into two subclasses, class I and II, based on amino acid sequence similarity. Blue-light photoreceptors are essential light detectors for the early development of plants. The amino acid sequence of the receptor is similar to sequences in the photolyases, although the receptor does not show photoreactivation activity. To investigate the functional divergence within the family, the amino acid sequences of the proteins were aligned. The alignment suggested that the recognition mechanisms of the cofactors and the substrate of class I CPD photolyases are different from those of class II CPD photolyases. Phylogenetic trees based on the alignment were reconstructed. Phylogenetic analysis suggests that the ancestral gene of the family had encoded CPD photolyase and that the gene duplication of the ancestral proteins had occurred at least eight times before the divergence between eubacteria and eukaryotes (Kanai, 1997).

Photolyase repairs ultraviolet (UV) damage to DNA by splitting the cyclobutane ring of the major UV photoproduct: the cis, syn-cyclobutane pyrimidine dimer (Pyr <> Pyr). The reaction is initiated by blue light and proceeds through long-range energy transfer, single electron transfer, and enzyme catalysis by a radical mechanism. The three-dimensional crystallographic structure of DNA photolyase from Escherichia coli is presented and the atomic model has been refined to an R value of 0.172 at 2.3 A resolution. The polypeptide chain of 471 amino acids is folded into an amino-terminal alpha/beta domain resembling dinucleotide binding domains and a carboxyl-terminal helical domain; a loop of 72 residues connects the domains. The light-harvesting cofactor 5,10-methenyltetrahydrofolylpolyglutamate (MTHF) binds in a cleft between the two domains. Energy transfer from MTHF to the catalytic cofactor flavin adenine dinucleotide (FAD) occurs over a distance of 16.8 A. The FAD adopts a U-shaped conformation between two helix clusters in the center of the helical domain and is accessible through a hole in the surface of this domain. Dimensions and polarity of the hole match those of a Pyr <> Pyr dinucleotide, suggesting that the Pyr <> Pyr "flips out" of the helix to fit into this hole, and that electron transfer between the flavin and the Pyr <> Pyr occurs over van der Waals contact distance (Park, 1995).

Employing UV-irradiated Drosophila melanogaster embryos as a source of material, three new proteins that selectively bind to UV-damaged DNA were identified and purified through to near homogeneity. These proteins, tentatively designated as D-DDB P1, P2 and P3, can be identified as different complex bands in a gel shift assay by using UV-irradiated TC-31 probe DNA. Analysis of the purified D-DDB P1 fraction by native or SDS-polyacrylamide gel electrophoresis and FPLC-Superose 6 gel filtration demonstrates that it is a monomer protein that is a 30 kDa polypeptide. The D-DDB P2 protein is a monopolypeptide with a molecular mass of 14 kDa. Both D-DDB P1 and P2 highly prefer binding to UV-irradiated DNA, and have almost no affinity for non-irradiated DNA. Gel shift assays with either UV-irradiated DNA probes demonstrates that D-DDB P1 may show a preference for binding to (6-4) photoproducts, while D-DDB P2 may prefer binding to pyrimidine dimers. Both these proteins require magnesium ions for binding. D-DDB P1 is an ATP-preferent protein. Also discussed is a possible role for these DNA-binding proteins in lesion recognition and DNA repair of UV-induced photo-products (Kai, 1995).

Animal-type photolyases have very limited sequence homology to microbial-type photolyases. Do animal and microbial enzymes have different or similar biochemical and photochemical properties? In particular, the chromophore/cofactor composition of animal photolyases is of special interest since neither the presence nor the nature of a second chromophore in these enzymes is known, in contrast to the microbial photolyases which contain FAD cofactor, and folate or deazaflavin as second chromophores. The Drosophila photolyase was produced in E. coli using the cloned gene. The enzyme contains FAD and folate and thus belongs in the folate class of enzymes but with an action spectrum peak at 420 nm (Kim, 1996).

Ultraviolet light (UV)-induced DNA damage can be repaired by DNA photolyase in a light-dependent manner. Two types of photolyase are known, one specific for cyclobutane pyrimidine dimers (CPD photolyase) and another specific for pyrimidine (6-4) pyrimidone photoproducts[(6-4)photolyase]. In contrast to the CPD photolyase, which has been detected in a wide variety of organisms, the (6-4)photolyase has been found only in Drosophila melanogaster. In the present study a gene encoding the Drosophila(6-4)photolyase was cloned, and the deduced amino acid sequence of the product was found to be similar to the CPD photolyase and to the blue-light photoreceptor of plants. A homolog of the Drosophila (6-4)photolyase gene was also cloned from human cells (Todo, 1996).

Highly purified D-DDB P1, an ultraviolet light (UV) damage-specific DNA-binding protein from Drosophila melanogaster, has been obtained from Drosophila Kc cells and found to be a nuclease. D-DDB P1 can selectively bind to pyrimidine (6-4) pyrimidone photoproducts, and in the presence of Mg++, D-DDB P1 can catalyze an incision immediately on the 3' and 5' sides of the (6-4) photoproduct site (Kai, 1998).

The (6-4)photoproduct DNA photolyase [(6-4)photolyase] repairs UV-induced pyrimidine (6-4) pyrimidone photoproduct [(6-4)photoproduct, pyr(6,4)pyr] in a light dependent manner. Drosophila (6-4)photolyase was purified to near homogeneity from Drosophila embryonic cells and is a 62 kDa protein as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified (6-4)photolyase repairs (6-4)photoproducts induced at 5'-CC-3' site (C[6,4]C) as well as T[6,4]T and T[6,4]C. Photoreactivation of (6-4)photoproduct constructed in M13 phage eliminates the replication block and abolishes induced mutagenesis in E. coli cells, suggesting that the (6-4)photolyase repairs the photoproduct to the unmodified form (Todo, 1998).

Yeast and many other organisms use nucleotide excision repair (NER) and photolyase in the presence of light (photoreactivation) to repair cyclobutane pyrimidine dimers (CPDs), a major class of DNA lesions generated by UV light. To study the role of photoreactivation at the chromatin level in vivo, yeast strains were used that contain minichromosomes (YRpTRURAP, YRpCS1) with well-characterized chromatin structures. The strains were either proficient (RAD1) or deficient (rad1 delta) in NER. In contrast to NER, photolyase rapidly repairs CPDs in non-nucleosomal regions, including promoters of active genes (URA3, HIS3, DED1) and in linker DNA between nucleosomes. CPDs in nucleosomes are much more resistant to photoreactivation. These results demonstrate a direct role of chromatin in modulation of a DNA repair process and an important role of photolyase in repair of damaged promoters with presumptive effects on gene regulation. In addition, photoreactivation provides an in vivo test for chromatin structure and stability. In active genes (URA3, HIS3), photolyase repairs the non-transcribed strand faster than the transcribed strand and can match fast removal of lesions from the transcribed strand by NER (transcription-coupled repair). Thus, the combination of both repair pathways ensures efficient repair of active genes (Suter, 1997).

The photolyase/blue-light photoreceptor family of proteins includes cyclobutane pyrimidine dimer photolyase, (6-4) photolyase and blue-light photoreceptors that were recently discovered in Arabidopsis thaliana, Sinapis alba and Chlamydomonas reinhardtii. Recently, two human genes, hCRY1 and hCRY2, have been identifed as members of this same family. The proteins encoded by these genes have no DNA repair activity and therefore are hypothesized to function in human blue-light response reactions. To identify downstream targets for these putative blue-light photoreceptors, interacting proteins were sought by the yeast two-hybrid method. The tetratricopeptide repeat protein 1, Tpr1, and the protein serine/threonine phosphatase 5 (PP5) that contains the TPR motif specifically interacts with hCRY2. The effect of the hCRY2-PP5 interaction on the protein phosphatase activity was investigated. hCRY2, but not the highly homologous (6-4) photolyase, is found to inhibit the phosphatase activity of PP5. This inhibition may be on the pathway of blue-light signal transduction reaction in humans (Zhao, 1997).

Specific responses to blue light are found throughout the biological kingdom. These responses (which in higher plants include phototropism, inhibition of hypocotyl elongation, and stomatal opening) are in many cases thought to be mediated by flavin-type photoreceptors. But no such blue-light photoreceptor has yet been identified or isolated, although blue-light responses in plants were reported by Darwin over a century ago, long before the discovery of the now relatively well characterized red/far-red light photoreceptor, phytochrome. Here the isolation of a gene corresponding to the HY4 locus of Arabidopsis thaliana is described. The hy4 mutant is one of several mutants that are selectively insensitive to blue light during the blue-light-dependent inhibition of hypocotyl elongation response, which suggests that they lack an essential component of the cryptochrome-associated light-sensing pathway. The HY4 gene, isolated by gene tagging, encodes a protein with significant homology to microbial DNA photolyases.Because photolyases comprise a rare class of flavoproteins that catalyse blue-light-dependent reactions, the protein encoded by HY4 has a structure consistent with that of a flavin-type blue-light photoreceptor (Ahmad, 1993).

A more recent paper by Ahmad (1995) reports the characterization of novel mutations within the Arabidopsis thaliana HY4 gene, which has previously been shown to encode a protein (CRY1) with characteristics of a blue-light photoreceptor. Several point mutations were identified within the amino-terminal domain of CRY1: this region of CRY1 has high homology to photolyase and is likely to be involved in blue-light-mediated electron transfer. Mutations were found within the region of homology to the known chromophore binding domains of photolyase. Point mutations within the 200 amino acid carboxy-terminal extension distinguishing CRY1 from photolyase, likewise disrupt function of the protein. CRY1 was originally defined as the photoreceptor responsible for blue-light-mediated inhibition of hypocotyl elongation and it is now reported that anthocyanin accumulation in germinating seedlings is an additional phenotype under the control of this photoreceptor. This is shown to be mediated in part by modulation of mRNA levels of chalcone synthase, one of the anthocyanin biosynthetic enzymes. The effect of the novel mutations on both inhibition of hypocotyl elongation and anthocyanin biosynthesis have been evaluated: mutations with less severe effects on hypocotyl elongation show a similarly reduced effect on anthocyanin biosynthesis. These results are consistent with the cryptochrome photoreceptor mediating multiple regulatory pathways by the same primary mode of action (Ahmad, 1995).

Blue-light responses in higher plants are mediated by specific photoreceptors, which are thought to be flavoproteins; one such flavin-type blue-light receptor, CRY1 (for cryptochrome), which mediates inhibition of hypocotyl elongation and anthocyanin biosynthesis, has recently been characterized. Prompted by classical photobiological studies suggesting possible co-action of the red/far-red absorbing photoreceptor phytochrome with blue-light photoreceptors in certain plant species, the role of phytochrome in CRY1 action in Arabidopsis was investigated. The activity of the CRY1 photoreceptor can be substantially altered by manipulating the levels of active phytochrome (Pfr) with red or far-red light pulses subsequent to blue-light treatments. Furthermore, analysis of severely phytochrome-deficient mutants shows that CRY1-mediated blue-light responses are considerably reduced, even though Western blots confirm that levels of CRY1 photoreceptor are unaffected in these phytochrome-deficient mutant backgrounds. It is concluded that CRY1-mediated inhibition of hypocotyl elongation and anthocyanin production requires active phytochrome for full expression, and that this requirement can be supplied by low levels of either phyA or phyB (Ahmad, 1997).

A cDNA from Arabidopsis thaliana similar to microbial photolyase genes, and designated AT-PHH1, was isolated using a photolyase-like cDNA from Sinapsis alba (SA-PHR1) as a probe. Multiple isolations yielded only PHH1 cDNAs, and a few blue-light-receptor CRY1 (HY4) cDNAs (also similar to microbial photolyase genes), suggesting the absence of any other highly similar Arabidopsis genes. The AT-PHH1 and SA-PHR1 cDNA sequences predict 89% identity at the protein level, except for an AT-PHH1 C-terminal extension (111 amino acids), also not seen in microbial photolyases. AT-PHH1 and CRY1 show less similarity, including respective C-terminal extensions that are themselves mostly dissimilar. Analysis of fifteen AT-PHH1 genomic isolates reveals a single gene, with three introns in the coding sequence and one in the 5'-untranslated leader. Full-length AT-PHH1, and both AT-PHH1 and AT-PHH1 delta C-513 (truncated to be approximately the size of microbial photolyase genes) cDNAs, were overexpressed, respectively, in yeast and Escherichia coli mutants hypersensitive to ultraviolet light. The absence of significant effects on resistance suggests either that any putative AT-PHH1 DNA repair activity requires cofactors/chromophores not present in yeast or E. coli, or that AT-PHH1 encodes a blue-light/ultraviolet-A receptor rather than a DNA repair protein (Hoffman, 1996).

Phototropism (bending toward the light) is one of the best known plant tropic responses. Despite being reported by Darwin and others over a century ago to be specifically under the control of blue light, the photoreceptors mediating phototropism have remained unknown. A blue-light photoreceptor from Arabidopsis, named CRY1 for cryptochrome 1 has been characterized; this photoreceptor is a flavoprotein that mediates numerous blue-light-dependent responses. In Arabidopsis, HY4 (the gene encoding CRY1) is a member of a small gene family that also encodes a related photoreceptor, CRY2, which shares considerable functional overlap with CRY1. Mutant plants lacking both the CRY1 and the CRY2 blue-light photoreceptors are deficient in the phototropic response. Transgenic Arabidopsis plants overexpressing CRY1 or CRY2 show enhanced phototropic curvature. It is concluded that cryptochrome is one of the photoreceptors mediating phototropism in plants (Ahmad, 1998a).

A blue light (cryptochrome) photoreceptor from Arabidopsis, cry1, has been identified recently and shown to mediate a number of blue light-dependent phenotypes. Similar to phytochrome, the cryptochrome photoreceptors are encoded by a gene family of homologous members with considerable amino acid sequence similarity within the N-terminal chromophore binding domain. The two members of the Arabidopsis cryptochrome gene family (CRY1 and CRY2) overlap in function, but their proteins differ in stability: cry2 is rapidly degraded under light fluences (green, blue, and UV) that activate the photoreceptor, but cry1 is not. It is demonstrated by overexpression in transgenic plants of cry1 and cry2 fusion constructs that their domains are functionally interchangeable. Hybrid receptor proteins mediate functions similar to cry1 and include inhibition of hypocotyl elongation and blue light-dependent anthocyanin accumulation; differences in activity appear to be correlated with differing protein stability. Because cry2 accumulates to high levels under low-light intensities, it may have greater significance in wild-type plants under conditions when light is limited (Ahmad, 1998b).

Plants have at least two major photosensory receptors: phytochrome (absorbing primarily red/far-red light) and cryptochrome (absorbing blue/UV-A light); considerable physiological and genetic evidence suggests some form of communication or functional dependence between the receptors. Arabidopsis CRY1 and CRY2 (cryptochrome) are substrates for phosphorylation by a phytochrome A-associated kinase activity. Several mutations within the CRY1 C terminus lead to reduced phosphorylation by phytochrome preparations in vitro. Yeast two-hybrid interaction studies using expressed C-terminal fragments of CRY1 and phytochrome A from Arabidopsis confirm a direct physical interaction between both photoreceptors. In vivo labeling studies and specific mutant alleles of CRY1, which interfere with the function of phytochrome, suggest the possible relevance of these findings in vivo (Ahmad, 1998c).

Cryptochromes are a group of flavin-type blue light receptors that regulate plant growth and development. The function of Arabidopsis cryptochrome 2 in the early photomorphogenesis of seedlings was studied by using transgenic plants overexpressing CRY2 protein, and cry2 mutant plants accumulating no CRY2 protein. Cryptochrome 2 mediates blue light-dependent inhibition of hypocotyl elongation and stimulation of cotyledon opening under low intensities of blue light. In contrast to CRY1, the expression of CRY2 is rapidly down-regulated by blue light in a light-intensity dependent manner, which provides a molecular mechanism to explain at least in part that cryptochrome 2 functions primarily under low light during the early development of seedlings (Lin, 1998).

The shift in plants from vegetative growth to floral development is regulated by red-far-red light receptors (phytochromes) and blue-ultraviolet A light receptors (cryptochromes). A mutation in the Arabidopsis thaliana CRY2 gene encoding a blue-light receptor apoprotein (CRY2) is allelic to the late-flowering mutant, fha. Flowering in cry2/fha mutant plants is only incompletely responsive to photoperiod. Cryptochrome 2 (cry2) is a positive regulator of the flowering-time gene CO, the expression of which is regulated by photoperiod. Analysis of flowering in cry2 and phyB mutants in response to different wavelengths of light indicates that flowering is regulated by the antagonistic actions of phyB and cry2 (Guo, 1998).

Cryptochrome blue light photoreceptors share sequence similarity to photolyases, flavoproteins that mediate light-dependent DNA repair. However, cryptochromes lack photolyase activity and are characterized by distinguishing C-terminal domains. The signaling mechanism of Arabidopsis cryptochrome is mediated through the C terminus. On fusion with beta-glucuronidase (GUS), both the Arabidopsis CRY1 C-terminal domain (CCT1) and the CRY2 C-terminal domain (CCT2) mediate a constitutive light response. This constitutive photomorphogenic (COP) phenotype is not observed for mutants of cct1 corresponding to previously described cry1 alleles. It is proposed that the C-terminal domain of Arabidopsis cryptochrome is maintained in an inactive state in the dark. Irradiation with blue light relieves this repression, presumably through an intra- or intermolecular redox reaction mediated through the flavin bound to the N-terminal photolyase-like domain (Yang, 2000).

It has been assumed that cryptochromes function in a manner analogous to photolyases, mediating a light-driven redox reaction. Such a reaction could be either intermolecular or intramolecular. In the simplest form of the 'intermolecular model', cryptochrome activates a signaling partner, bound to the C teminus. In this model, which is very similar to the mode of action of photolyases, the C terminus undergoes no change as a result of irradiation. It is the bound signaling partner that undergoes a redox-induced change, with the function of CCT simply being to bind this partner. From the results that have been presented here, this model is wrong, as the 'isolated' CCT clearly displays properties distinct from those associated with CRY present in dark-grown seedlings. Furthermore, since these properties of the CCT are strikingly similar to those of light-activated CRY, and are not displayed by mutants of CCT, it is presumed that they are physiologically meaningful (Yang, 2000).

An alternative version of the 'intermolecular model', in keeping with the observations made in this study, postulates that upon light-induced activation, a regulatory molecule (Y) is released from its complex with CRY, thus exposing CCT and enabling binding to the signaling partner X. This model is similar in some ways to the regulation of protein kinase A, or glucocorticoid receptor, where ligand binding results in a change in conformation and release of regulatory subunits. For this model to hold, CRY would need to be fully associated in the dark with such a presumptive negative regulatory subunit (Yang, 2000).

In contrast to photolyases, cryptochrome may function through an intramolecular redox reaction, resulting in a change in the chemical properties of the CCT. If the catalytic form of the flavin in CRY is in the form of FADH-, as it is in photolyases, then the most likely acceptor for a light-induced redox change is a cysteine residue. CCT1 does not contain any cysteine residues, and therefore such an intramolecular redox reaction would need to be restricted to the N-terminal photolyase-like domain of CRY1. Alternatively, the catalytically active form of the flavin for CRY may be FAD or the semiquinone (FADH.), which for CRY1 has been demonstrated to be surprisingly stable. The significance of this point is that the flavin in either of these redox states may act as an electron acceptor, and a tryptophan residue (for example) could serve as the corresponding donor (Yang, 2000).

Somewhat irrespective of the details of the light-induced activation of CCT, the data indicate that CRY function is achieved through a signaling event mediated through the C terminus. This conclusion follows from the findings concerning the activity of the GUS-CCT transgenes and is in keeping with observations regarding mutant cry1 alleles containing lesions within the C terminus. This signaling likely involves interaction of CCT with another protein(s) and the identity of such a protein is obviously of interest (Yang, 2000).

Many of the properties of Arabidopsis seedlings expressing the CCT transgenes are reminiscent of mutants that affect either COP1 or the COP9 complex. Mutations in COP1, a zinc finger protein, result in short hypocotyls in the dark, and abnormal gene expression and chloroplast development, similar to those described here for the CCT-expressing plants. Whereas many alleles of cop1 are sterile, weak alleles are fertile and flower early, again similar in these respects to the CCT plants. Many of these properties of cop1 alleles are shared by mutants affecting other components of the COP9 signalosome, a multisubunit complex that has homologies to animal proteins and is believed to play a role in protein degradation. The COP9 complex is required for COP1 accumulation, which in turn negatively regulates photomorphogenesis. This latter step involves HY5, a bZIP DNA binding protein that physically interacts with COP1. HY5 levels are increased in the light, apparently a result of COP1 translocation from the nucleus to the cytoplasm (Yang, 2000 and references therein).

In view of the similarity of the mutant plants expressing CCT and both the COP1 mutants and mutants of the subunits of the COP9 complex, the following is considered as a likely explanation for the CCT phenotype: subsequent to the redox-mediated activation of the C terminus, it is proposed that CCT binds to one of four possible types of signaling partners. In the first option, CRY interacts in a negative manner with COP1, in this manner phenocopying loss-of-function cop1 alleles. In a similar manner, light-activated CRY may negatively interact with any one of the many COP9 signalosome subunits; again these interactions would phenocopy loss-of-function mutations affecting any of the subunits of this complex. Third, CRY may interact with signaling molecules downstream, or (fourth) upstream from the COP1 and the COP9 complex. In future studies, the details of the redox reaction and the identity of these CRY signaling partner(s) need to be identified. Furthermore, it will be of interest to see if the general properties described here for Arabidopsis CRY, are found to hold for animal cryptochromes, which, at least in the case of Drosophila CRY, also function as photoreceptors (Yang, 2000 and references therein).

Most organisms, from cyanobacteria to mammals, use circadian clocks to coordinate their activities with the natural 24-h light/dark cycle. The clock proteins of Drosophila and mammals exhibit striking homology but do not show similarity with clock proteins found so far from either cyanobacteria or Neurospora. Each of these organisms uses a transcriptionally regulated negative feedback loop in which the messenger RNA levels of the clock components cycle over a 24-h period. Proteins containing PAS domains are invariably found in at least one component of the characterized eukaryotic clocks. This study describes ADAGIO1 (ADO1), a gene of Arabidopsis thaliana that encodes a protein containing a PAS domain. A loss-of-function ado1 mutant is altered in both gene expression and cotyledon movement in circadian rhythmicity. Under constant white or blue light, the ado1 mutant exhibits a longer period than that of wild-type Arabidopsis seedlings, whereas under red light cotyledon movement and stem elongation are arrhythmic. Both yeast two-hybrid and in vitro binding studies show that there is a physical interaction between ADO1 and the photoreceptors CRY1 and phyB (phytochromes are red/far-red light-absorbing receptors). It is proposed that ADO1 is an important component of the Arabidopsis circadian system (Jarillo, 2001).

Cryptochromes (CRY) are blue light photoreceptors that mediate various light-induced responses in plants and animals. Arabidopsis CRY (CRY1 and CRY2) functions through negatively regulating constitutive photomorphogenic (COP) 1, a repressor of photomorphogenesis. Water evaporation and photosynthesis are regulated by the stomatal pores in plants, which are closed in darkness but open in response to blue light. There is evidence only for the phototropin blue light receptors (PHOT1 and PHOT2) in mediating blue light regulation of stomatal opening. This study reports previously uncharacterized role for Arabidopsis CRY and COP1 in the regulation of stomatal opening. Stomata of the cry1 cry2 double mutant showed reduced blue light response, whereas those of the CRY1-overexpressing plants showed hypersensitive response to blue light. In addition, stomata of the phot1 phot2 double mutant responded to blue light, but those of the cry1 cry2 phot1 phot2 quadruple mutant hardly responded. Strikingly, stomata of the cop1 mutant are constitutively open in darkness and stomata of the cry1 cry2 cop1 and phot1 phot2 cop1 triple mutants are open as wide as those of the cop1 single mutant under blue light. These results indicate that CRY functions additively with PHOT in mediating blue light-induced stomatal opening and that COP1 is a repressor of stomatal opening and likely acts downstream of CRY and PHOT signaling pathways (Mao, 2005).

Cryptochromes are blue light-sensing photoreceptors found in plants, animals, and humans. They are known to play key roles in the regulation of the circadian clock and in development. However, despite striking structural similarities to photolyase DNA repair enzymes, cryptochromes do not repair double-stranded DNA, and their mechanism of action is unknown. Recently, a blue light-dependent intramolecular electron transfer to the excited state flavin was characterized and proposed as the primary mechanism of light activation. The resulting formation of a stable neutral flavin semiquinone intermediate enables the photoreceptor to absorb green/yellow light (500-630 nm) in addition to blue light in vitro. This study demonstrates that Arabidopsis cryptochrome activation by blue light can be inhibited by green light in vivo consistent with a change of the cofactor redox state. Light-dependent changes in the cryptochrome1 (cry1) protein was further characterized in living cells, which match photoreduction of the purified cry1 in vitro. These experiments were performed using fluorescence absorption/emission and EPR on whole cells and thereby represent one of the few examples of the active state of a known photoreceptor being monitored in vivo. These results indicate that cry1 activation via blue light initiates formation of a flavosemiquinone signaling state that can be converted by green light to an inactive form. In summary, cryptochrome activation via flavin photoreduction is a reversible mechanism novel to blue light photoreceptors. This photocycle may have adaptive significance for sensing the quality of the light environment in multiple organisms (Bouly, 2007).

Zebrafish tissues and cells have the unusual feature of not only containing a circadian clock, but also being directly light-responsive. Several zebrafish genes are induced by light, but little is known about their role in clock resetting or the mechanism by which this might occur. This study shows that Cryptochrome 1a (Cry1a) plays a key role in light entrainment of the zebrafish clock. Intensity and phase response curves reveal a strong correlation between light induction of Cry1a and clock resetting. Overexpression studies show that Cry1a acts as a potent repressor of clock function and mimics the effect of constant light to 'stop' the circadian oscillator. Yeast two-hybrid analysis demonstrates that the Cry1a protein interacts directly with specific regions of core clock components, CLOCK and BMAL, blocking their ability to fully dimerize and transactivate downstream targets, providing a likely mechanism for clock resetting. A comparison of entrainment of zebrafish cells to complete versus skeleton photoperiods reveals that clock phase is identical under these two conditions. However, the amplitude of the core clock oscillation is much higher on a complete photoperiod, as are the levels of light-induced Cry1a. It is believed that Cry1a acts on the core clock machinery in both a continuous and discrete fashion, leading not only to entrainment, but also to the establishment of a high-amplitude rhythm and even stopping of the clock under long photoperiods (Tamai, 2007).