Cryptochromes are photolyase-like blue light receptors originally discovered in Arabidopsis but later found in other plants, microbes, and animals. Arabidopsis has two cryptochromes, CRY1 and CRY2, which mediate primarily blue light inhibition of hypocotyl elongation and photoperiodic control of floral initiation, respectively. In addition, cryptochromes also regulate over a dozen other light responses, including circadian rhythms, tropic growth, stomata opening, guard cell development, root development, bacterial and viral pathogen responses, abiotic stress responses, cell cycles, programmed cell death, apical dominance, fruit and ovule development, seed dormancy, and magnetoreception. Cryptochromes have two domains, the N-terminal PHR (Photolyase-Homologous Region) domain that bind the chromophore FAD (flavin adenine dinucleotide), and the CCE (CRY C-terminal Extension) domain that appears intrinsically unstructured but critical to the function and regulation of cryptochromes. Most cryptochromes accumulate in the nucleus, and they undergo blue light-dependent phosphorylation or ubiquitination. It is hypothesized that photons excite electrons of the flavin molecule, resulting in redox reaction or circular electron shuttle and conformational changes of the photoreceptors. The photoexcited cryptochrome are phosphorylated to adopt an open conformation, which interacts with signaling partner proteins to alter gene expression at both transcriptional and posttranslational levels and consequently the metabolic and developmental programs of plants.
Cryptochromes are photoreceptors that regulate entrainment of the circadian clock by light in plants and animals. They are related to DNA photolyases and have similar three-dimensional structures, characterized by a α/β domain and a helical domain and including a chromophore, flavin adenine dinucleotide.
Cryptochromes are photoreceptors that regulate entrainment by light of the circadian clock in plants and animals. They also act as integral parts of the central circadian oscillator in animal brains and as receptors controlling photomorphogenesis in response to blue or ultraviolet (UV-A) light in plants. Cryptochromes are probably the evolutionary descendents of DNA photolyases, which are light-activated DNA-repair enzymes, and are classified into three groups - plant cryptochromes, animal cryptochromes, and CRY-DASH proteins. Cryptochromes and photolyases have similar three-dimensional structures, characterized by an α/β domain and a helical domain. The structure also includes a chromophore, flavin adenine dinucleotide (FAD). The FAD-access cavity of the helical domain is the catalytic site of photolyases, and it is predicted also to be important in the mechanism of cryptochromes.
The photolyase/cryptochrome family of proteins are FAD-containing flavoproteins which carry out blue-light dependent functions including DNA repair, plant growth and development, and regulation of the circadian clock. In addition to FAD, many members of the family contain a second chromophore which functions as a photoantenna, harvesting light and transferring the excitation energy to FAD and thus increasing the efficiency of the system. The second chromophore is methenyltetrahydrofolate (MTHF) in most photolyases characterized to date and FAD, FMN, or 5-deazariboflavin in others. To date no second chromophore has been identified in cryptochromes. Drosophila contains 3 members of the cryptochrome/photolyase family: cyclobutane pyrimidine dimer (CPD) photolyase, (6-4) photoproduct photolyase, and cryptochrome. We developed an expression system capable of incorporating all known second chromophores into the cognate cryptochrome/photolyase family members. Using this system we demonstrate that Drosophila CPD photolyase and (6-4) photolyase employ 5-deazariboflavin as their second chromophore but Drosophila cryptochrome, which is evolutionarily closer to (6-4) photolyase than the CPD photolyase, lacks a second chromophore.
Photolyases and cryptochromes are evolutionarily related flavoproteins with distinct functions. While photolyases can repair UV-induced DNA lesions in a light-dependent manner, cryptochromes regulate growth, development and the circadian clock in plants and animals. Here we report about two photolyase-related proteins, named PhrA and PhrB, found in the phytopathogen Agrobacterium tumefaciens. PhrA belongs to the class III cyclobutane pyrimidine dimer (CPD) photolyases, the sister class of plant cryptochromes, while PhrB belongs to a new class represented in at least 350 bacterial organisms. Both proteins contain flavin adenine dinucleotide (FAD) as a primary catalytic cofactor, which is photoreduceable by blue light. Spectral analysis of PhrA confirmed the presence of 5,10-methenyltetrahydrofolate (MTHF) as antenna cofactor. PhrB comprises also an additional chromophore, absorbing in the short wavelength region but its spectrum is distinct from known antenna cofactors in other photolyases. Homology modeling suggests that PhrB contains an Fe-S cluster as cofactor which was confirmed by elemental analysis and EPR spectroscopy. According to protein sequence alignments the classical tryptophan photoreduction pathway is present in PhrA but absent in PhrB. Although PhrB is clearly distinguished from other photolyases including PhrA it is, like PhrA, required for in vivo photoreactivation. Moreover, PhrA can repair UV-induced DNA lesions in vitro. Thus, A. tumefaciens contains two photolyase homologs of which PhrB represents the first member of the cryptochrome/photolyase family (CPF) that contains an iron-sulfur cluster.
Cryptochromes are FAD-based blue-light photoreceptors that regulate growth and development in plants and the circadian clock in animals. Arabidopsis thaliana and humans possess two cryptochromes. Recently, it was found that Arabidopsis cryptochrome 1 (AtCry1) binds ATP and exhibits autokinase activity that is simulated by blue light. Similarly, it was reported that human cryptochrome 1 (HsCry1) exhibited autophosphorylation activity under blue light. To test the generality of light stimulated kinase function of cryptochromes, we purified AtCry1, AtCry2, HsCry1 and HsCry2 and probed them for kinase activity under a variety of conditions. We find that AtCry1, which contains near stoichiometric amount of FAD and human HsCry1 and HsCry2, which contain only trace amounts of FAD have autokinase activity but AtCry2, which also contains stoichiometric amounts of FAD does not. Finally, we find that the kinase activity of AtCry1 is not significantly affected by light or the redox status of the flavin cofactor.
Arabidopsis cryptochrome 2 (CRY2) is a blue-light receptor mediating blue-light inhibition of hypocotyl elongation and photoperiodic promotion of floral initiation. CRY2 is a constitutive nuclear protein that undergoes blue-light-dependent phosphorylation, ubiquitination, photobody formation, and degradation in the nucleus, but the relationship between these blue-light-dependent events remains unclear. It has been proposed that CRY2 phosphorylation triggers a conformational change responsible for the subsequent ubiquitination and photobody formation, leading to CRY2 function and/or degradation. We tested this hypothesis by a structure-function study, using mutant CRY2–GFP fusion proteins expressed in transgenic Arabidopsis. We show that changes of lysine residues of the NLS (Nuclear Localization Signal) sequence of CRY2 to arginine residues partially impair the nuclear importation of the CRY2K541R and CRY2K554/5R mutant proteins, resulting in reduced phosphorylation, physiological activities, and degradation in response to blue light. In contrast to the wild-type CRY2 protein that forms photobodies exclusively in the nucleus, the CRY2K541R and CRY2K554/5R mutant proteins form protein bodies in both the nucleus and cytosol in response to blue light. These results suggest that photoexcited CRY2 molecules can aggregate to form photobody-like structure without the nucleus-dependent protein modifications or the association with the nuclear CRY2-interacting proteins. Taken together, the observation that CRY2 forms photobodies markedly faster than CRY2 phosphorylation in response to blue light, we hypothesize that the photoexcited cryptochromes form oligomers, preceding other biochemical changes of CRY2, to facilitate photobody formation, signal amplification, and propagation, as well as desensitization by degradation.
protein phosphorylation; signal transduction; fluorescence imaging; protein degradation; photobody
Cryptochrome 2 (CRY2) is a blue/UV-A light receptor that regulates light inhibition of cell elongation and photoperiodic promotion of floral initiation in Arabidopsis. We and others have previously shown that CRY2 is a nuclear protein that regulates gene expression to affect plant development. We also showed that CRY2 is phosphorylated in response to blue light and the phosphorylated CRY2 is most likely active and degraded in blue light. Given that protein translation (and probably chromophore attachment) takes place in the cytosol and that a photoreceptor would absorb photon instantaneously, it would be interesting to know where those inter-connected events occur in the cell. Our results showed that freshly synthesized CRY2 photoreceptor is inactive in the cytosol although it may be photon-excited, it is imported into the nucleus where the photoreceptor is phosphorylated, performs its function, becomes ubiquitinated, and eventually gets degraded (Fig. 1).1 To our knowledge, this is the first example in any organism that a photoreceptor is shown to complete its post-translational life cycle in a single subcellular compartment.
blue light; cryptochrome; ubiquitination; phosphorylation; Arabidopsis
The Cryptochrome/Photolyase (CRY/PL) family of photoreceptors mediates adaptive responses to UV and blue light exposure in all kingdoms of life 1; 2; 3; 4; 5. Whereas PLs function predominantly in DNA repair of cyclobutane pyrimidine dimers (CPDs)and 6-4 photolesions caused by UV radiation, CRYs transduce signals important for growth, development, magnetosensitivity and circadian clocks1; 2; 3; 4; 5. Despite these diverse functions, PLs/CRYs preserve a common structural fold, a dependence on flavin adenine dinucleotide (FAD) and an internal photoactivation mechanism3; 6. However, members of the CRY/PL family differ in the substrates recognized (protein or DNA), photochemical reactions catalyzed and involvement of an antenna cofactor. It is largely unknown how the animal CRYs that regulate circadian rhythms act on their substrates. CRYs contain a variable C-terminal tail that appends the conserved PL homology domain (PHD) and is important for function 7; 8; 9; 10; 11; 12. Herein, we report a 2.3 Å resolution crystal structure of Drosophila CRY with an intact C-terminus. The C-terminal helix docks in the analogous groove that binds DNA substrates in PLs. Conserved Trp536 juts into the CRY catalytic center to mimic PL recognition of DNA photolesions. The FAD anionic semiquinone found in the crystals assumes a conformation to facilitate restructuring of the tail helix. These results help reconcile the diverse functions of the CRY/PL family by demonstrating how conserved protein architecture, and photochemistry can be elaborated into a range of light-driven functions.
Recombinant cryptochrome 3 from A. thaliana with FAD and MTHF cofactors has been crystallized using the hanging-drop vapour-diffusion technique in the orthorhombic space group P212121 and X-ray diffraction data were collected to 1.9 Å resolution.
Cryptochromes are flavoproteins which serve as blue-light receptors in plants, animals, fungi and prokaryotes and belong to the same protein family as the catalytically active DNA photolyases. Cryptochrome 3 from the plant Arabidopsis thaliana (cry3; 525 amino acids, 60.7 kDa) is a representative of the novel cryDASH subfamily of UV-A/blue-light receptors and has been expressed as a mature FAD-containing protein in Escherichia coli without the signal sequence that directs the protein into plant organelles. The purified cryptochrome was found to be complexed to methenyltetrahydrofolate as an antenna pigment. Crystals of the cryptochrome–antenna pigment complex were obtained by vapour diffusion and display orthorhombic symmetry, with unit-cell parameters a = 76.298, b = 116.782, c = 135.024 Å. X-ray diffraction data were collected to 1.9 Å resolution using synchrotron radiation. The asymmetric unit comprises a cry3 dimer, the physiological role of which remains to be elucidated.
cryptochrome 3; light receptors
Cryptochromes are a class of flavoprotein blue-light signaling receptors found in plants, animals, and humans that control plant development and the entrainment of circadian rhythms. In plant cryptochromes, light activation is proposed to result from photoreduction of a protein-bound flavin chromophore through intramolecular electron transfer. However, although similar in structure to plant cryptochromes, the light-response mechanism of animal cryptochromes remains entirely unknown. To complicate matters further, there is currently a debate on whether mammalian cryptochromes respond to light at all or are instead activated by non–light-dependent mechanisms. To resolve these questions, we have expressed both human and Drosophila cryptochrome proteins to high levels in living Sf21 insect cells using a baculovirus-derived expression system. Intact cells are irradiated with blue light, and the resulting cryptochrome photoconversion is monitored by fluorescence and electron paramagnetic resonance spectroscopic techniques. We demonstrate that light induces a change in the redox state of flavin bound to the receptor in both human and Drosophila cryptochromes. Photoreduction from oxidized flavin and subsequent accumulation of a semiquinone intermediate signaling state occurs by a conserved mechanism that has been previously identified for plant cryptochromes. These results provide the first evidence of how animal-type cryptochromes are activated by light in living cells. Furthermore, human cryptochrome is also shown to undergo this light response. Therefore, human cryptochromes in exposed peripheral and/or visual tissues may have novel light-sensing roles that remain to be elucidated.
Vision in animals is generally associated with light-sensitive rhodopsin pigments located in the eyes. However, animals ranging from flies to humans also possess ancient visual receptors known as cryptochromes in multiple cell types. In this work, we study the mechanism of light sensing in two representative animal cryptochromes: a light-sensitive Drosophila cryptochrome (Dmcry) and a presumed light-insensitive mammalian cryptochrome from humans (Hscry1). We expressed recombinant cryptochromes to high levels in living cells, irradiated the cells with blue light, and analyzed the proteins' response to irradiation with electron paramagnetic resonance and fluorescence spectroscopic techniques. Photoreduction of protein-bound oxidized FAD cofactor to its radical form emerged as the primary cryptochrome photoreaction in living cells, and was correlated with a light-sensitive biological response in whole organisms. These results indicate that both Dmcry and Hscry1 are capable of undergoing similar light-driven reactions and suggest the possibility of an as-yet unknown photo-perception role for human cryptochromes in tissues exposed to light.
Cryptochromes are blue-light-absorbing receptors found in plants, animals, and humans. In mammals, they are not thought to respond to light, but this study demonstrates contrary evidence that indeed, human cryptochromes undergo a photochemical transformation in response to light.
Photolyase/cryptochrome family is a large family of flavoproteins that encompasses DNA repair proteins, photolyases; and cryptochromes that regulate blue-light dependent growth and development in plants, and light-dependent and light-independent circadian clock-setting in animals. Phylogenetic analysis has revealed a new branch of the family which co-segregates with plant cryptochromes. Here we describe the isolation and characterization of a member of this family named Type III photolyase, from Caulobacter crescentus. Spectroscopic analysis shows that the enzyme contains both the methenyl-tetrahydrofolate photoantenna and the FAD catalytic cofactor. Biochemical analysis shows that it is a bona fide photolyase that repairs cyclobutane pyrimidine dimers. Mutation of an active site Trp to Arg disrupts FAD binding with no measurable effect on MTHF binding. Using enzyme preparations that contain either or both chromophores we were able to determine the efficiency and rate of energy transfer from MTHF to FAD. Photolyase/cryptochrome family is a large family of flavoproteins that encompasses DNA repair proteins, photolyases; and cryptochromes that regulate blue-light dependent growth and development in plants, and light-dependent and light-independent circadian clock-setting in animals. Phylogenetic analysis has revealed a new branch of the family which co-segregates with plant cryptochromes. Here we describe the isolation and characterization of a member of this family named Type III photolyase, from Caulobacter crescentus. Spectroscopic analysis shows that the enzyme contains both the methenyl-tetrahydrofolate photoantenna and the FAD catalytic cofactor. Biochemical analysis shows that it is a bona fide photolyase that repairs cyclobutane pyrimidine dimers. Mutation of an active site Trp to Arg disrupts FAD binding with no measurable effect on MTHF binding. Using enzyme preparations that contain either or both chromophores we were able to determine the efficiency and rate of energy transfer from MTHF to FAD.
Cryptochromes (CRYs) are photoreceptors mediating developmental responses to blue light throughout the life of plants. Function and signal transduction of CRYs in photomorphogenesis have been well characterized in Arabidopsis. Studies on rice CRYs demonstrate that monocots CRYs may function similarly to their Arabidopsis counterparts. However, there is inconsistency in subcellular localization of CRYs in different species and little has been known about the effects of environmental cues on CRYs except for light. We recently reported that TaCRY1a of monocot wheat displays a light-responsive nucleocytoplasmic shuttling pattern similar to Arabidopsis CRY1 but differs from AtCRY1 and OsCRY1 by containing nuclear localization domains in both its N and C termini and the sequence for nuclear export in its N-terminal domain. TaCRY1a and TaCRY2 are transcriptionally regulated by osmotic stress/ABA and overexpression of TaCRY1a-GFP and TaCRY2-GFP led to higher sensitivity to high salinity, osmotic stress and ABA treatment. Mining wheat EST database provided additional clues for CRY's involvement in pathways apart from photomorphogenesis.
cryptochrome; signal transduction; stress; subcellular localization; wheat
Cryptochromes are blue-light receptors that have presumably evolved from the DNA photolyase protein family, and the genomes of many organisms contain genes for both types of molecules. Both protein structures resemble each other, which suggests that light control and light protection share a common ancient origin. In the genome of the filamentous fungus Aspergillus nidulans, however, only one cryptochrome/photolyase-encoding gene, termed cryA, was identified. Deletion of the cryA gene triggers sexual differentiation under inappropriate culture conditions and results in up-regulation of transcripts encoding regulators of fruiting body formation. CryA is a protein whose N- and C-terminal synthetic green fluorescent protein fusions localize to the nucleus. CryA represses sexual development under UVA350-370 nm light both on plates and in submerged culture. Strikingly, CryA exhibits photorepair activity as demonstrated by heterologous complementation of a DNA repair-deficient Escherichia coli strain as well as overexpression in an A. nidulans uvsBΔ genetic background. This is in contrast to the single deletion cryAΔ strain, which does not show increased sensitivity toward UV-induced damage. In A. nidulans, cryA encodes a novel type of cryptochrome/photolyase that exhibits a regulatory function during light-dependent development and DNA repair activity. This represents a paradigm for the evolutionary transition between photolyases and cryptochromes.
Cryptochromes (Cry) have been suggested to form the basis of light-dependent magnetic compass orientation in birds. However, to function as magnetic compass sensors, the cryptochromes of migratory birds must possess a number of key biophysical characteristics. Most importantly, absorption of blue light must produce radical pairs with lifetimes longer than about a microsecond. Cryptochrome 1a (gwCry1a) and the photolyase-homology-region of Cry1 (gwCry1-PHR) from the migratory garden warbler were recombinantly expressed and purified from a baculovirus/Sf9 cell expression system. Transient absorption measurements show that these flavoproteins are indeed excited by light in the blue spectral range leading to the formation of radicals with millisecond lifetimes. These biophysical characteristics suggest that gwCry1a is ideally suited as a primary light-mediated, radical-pair-based magnetic compass receptor.
Cryptochromes are blue light receptors that mediate light regulation of gene expression in all major evolution lineages, but the molecular mechanism underlying cryptochrome signal transduction remains not fully understood [1, 2]. It has been reported that cryptochromes suppress activity of the multifunctional E3 ubiquitin ligase CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1) to regulate gene expression in response to blue light [3, 4]. But how plant cryptochromes mediate light suppression of COP1 activity remains unclear. We report here that Arabidopsis CRY2 (cryptochrome 2) undergoes blue light-dependent interaction with the COP1-interacting protein SUPPRESSOR OF PHYTOCHROME A 1 (SPA1) [5, 6]. We demonstrate that SPA1 acts genetically downstream from CRY2 to mediate blue light suppression of the COP1-dependent proteolysis of the flowering-time regulator CONSTANS (CO) [7, 8]. We further show that blue light-dependent CRY2-SPA1 interaction stimulates CRY2-COP1 interaction. These results reveal for the first time a wavelength-specific mechanism by which a cryptochrome photoreceptor mediates light regulation of protein degradation to modulate developmental timing in Arabidopsis.
Cryptochromes are blue light photoreceptors involved in development and circadian clock regulation. They are found in both eukaryotes and prokaryotes as light sensors. Long Hypocotyl in Far-Red 1 (HFR1) has been identified as a positive regulator and a possible transcription factor in both blue and far-red light signaling in plants. However, the gene targets that are regulated by HFR1 in cryptochrome 1 (cry1)-mediated blue light signaling have not been globally addressed. We examined the transcriptome profiles in a cry1- and HFR1-dependent manner in response to 1 hour of blue light. Strikingly, more than 70% of the genes induced by blue light in an HFR1-dependent manner were dependent on cry1, and vice versa. High overrepresentation of W-boxes and OCS elements were found in these genes, indicating that this strong cry1 and HFR1 co-regulation on gene expression is possibly through these two cis-elements. We also found that cry1 was required for maintaining the HFR1 protein level in blue light, and that the HFR1 protein level is strongly correlated with the global gene expression pattern. In summary, HFR1, which is fine-tuned by cry1, is crucial for regulating global gene expression in cry1-mediated early blue light signaling, especially for the function of genes containing W-boxes and OCS elements.
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.
Cryptochromes (CRYs) are flavoproteins that are known as blue light photoreceptors in many organisms. Recently, genome sequences from a variety of algae became available. Functional characterizations of animal-like CRYs from Oestreococcus tauri, Chlamydomonas reinhardtii and Phaeodactylum tricornutum highlighted novel functions and properties. As arising from studies in fungi, certain algal CRYs of the “cryptochrome photolyase family” (PtCPF1, OtCPF1) have dual or even triple functions. They are involved in blue light perception and/or in the circadian clock and are able to repair DNA damages. On the other hand, the animal-like aCRY from C. reinhardtii is not only acting as sensory blue light- but also as sensory red light receptor thus expanding our current view of flavoproteins in general and CRYs in particular. The observed broad spectral response points to the neutral radical state of flavin, which is assumed to be the dark form in aCRY in contrast to the plant CRYs.
Chlamydomonas reinhardtii; cryptochrome; blue light photoreceptor; red light photoreceptor; photolyase; flavoprotein
In plants and animals, cryptochromes function as either photoreceptors or circadian clock components. We have examined the cryptochrome from the filamentous fungus Neurospora crassa and demonstrate that Neurospora cry encodes a DASH-type cryptochrome that appears capable of binding flavin adenine dinucleotide (FAD) and methenyltetrahydrofolate (MTHF). The cry transcript and CRY protein levels are strongly induced by blue light in a wc-1-dependent manner, and cry transcript is circadianly regulated, with a peak abundance opposite in phase to frq. Neither deletion nor overexpression of cry appears to perturb the free-running circadian clock. However, cry disruption knockout mutants show a small phase delay under circadian entrainment. Using electrophoretic mobility shift assays (EMSA), we show that CRY is capable of binding single- and double-stranded DNA (ssDNA and dsDNA, respectively) and ssRNA and dsRNA. Whole-genome microarray experiments failed to identify substantive transcriptional regulatory activity of cry under our laboratory conditions.
Cryptochromes (CRYs) are flavoproteins sharing high homology with photolyases. Some of them have function(s) including transcription regulation in the circadian clock oscillation, blue-light photoreception for resetting the clock phase, and light-dependent magnetoreception. Vertebrates retain multiple sets of CRY or CRY-related genes, but their functions are yet unclear especially in the lower vertebrates. Although CRYs and the other circadian clock components have been extensively studied in the higher vertebrates such as mice, only a few model species have been studied in the lower vertebrates. In this study, we identified two CRYs, XtCRY1 and XtCRY2 in Xenopus tropicalis, an excellent experimental model species. Examination of tissue specificity of their mRNA expression by real-time PCR analysis revealed that both the XtCRYs showed extremely high mRNA expression levels in the ovary. The mRNA levels in the ovary were about 28-fold (XtCry1) and 48-fold (XtCry2) higher than levels in the next abundant tissues, the retina and kidney, respectively. For the functional analysis of the XtCRYs, we cloned circadian positive regulator XtCLOCK and XtBMAL1, and found circadian enhancer E-box in the upstream of XtPer1 gene. XtCLOCK and XtBMAL1 exhibited strong transactivation from the XtPer1 E-box element, and both the XtCRYs inhibited the XtCLOCK:XtBMAL1-mediated transactivation, thereby suggesting this element to drive the circadian transcription. These results revealed a conserved main feedback loop in the X. tropicalis circadian clockwork and imply a possible physiological importance of CRYs in the ovarian functions such as synthesis of steroid hormones and/or control of estrus cycles via the transcription regulation.
Cryptochromes (CRYs) are a class of flavoprotein blue-light signaling receptors found in plants and animals, and they control plant development and the entrainment of circadian rhythms. They also act as integral parts of the central circadian oscillator in humans and other animals. In mammals, the CLOCK-BMAL1 heterodimer activates transcription of the Per and Cry genes as well as clock-regulated genes. The PER2 proteins interact with CRY and CKIε, and the resulting ternary complexes translocate into the nucleus, where they negatively regulate the transcription of Per and Cry core clock genes and other clock-regulated output genes. Recent studies have indicated that the extended C-termini of the mammalian CRYs, as compared to photolyase proteins, interact with PER proteins.
We identified a region on mCRY2 (between residues 493 and 512) responsible for direct physical interaction with mPER2 by mammalian two-hybrid and co-immunoprecipitation assays. Moreover, using oligonucleotide-based degenerate PCR, we discovered that mutation of Arg-501 and Lys-503 of mCRY2 within this C-terminal region totally abolishes interaction with PER2.
Our results identify mCRY2 amino acid residues that interact with the mPER2 binding region and suggest the potential for rational drug design to inhibit CRYs for specific therapeutic approaches.
The photolyases, DNA repair enzymes that use visible and long-wavelength UV light to repair cyclobutane pyrimidine dimers (CPDs) created by short-wavelength UV, belong to the larger photolyase-cryptochrome gene family. Cryptochromes (UVA-blue light photoreceptors) lack repair activity, and sensory and regulatory roles have been defined for them in plants and animals. Evolutionary considerations indicate that cryptochromes diverged from CPD photolyases before the emergence of eukaryotes. In prokaryotes and lower eukaryotes, some photolyases might have photosensory functions. phr1 codes for a class I CPD photolyase in Trichoderma atroviride. phr1 is rapidly induced by blue and UVA light, and its photoinduction requires functional blue light regulator (BLR) proteins, which are White Collar homologs in Trichoderma. Here we show that deletion of phr1 abolished photoreactivation of UVC (200 to 280 nm)-inhibited spores and thus that PHR1 is the main component of the photorepair system. The 2-kb 5′ upstream region of phr1, with putative light-regulated elements, confers blue light regulation on a reporter gene. To assess phr1 photosensory function, fluence response curves of this light-regulated promoter were tested in null mutant (Δphr1) strains. Photoinduction of the phr1 promoter in Δphr1 strains was >5-fold more sensitive to light than that in the wild type, whereas in PHR1-overexpressing lines the sensitivity to light increased about 2-fold. Our data suggest that PHR1 may regulate its expression in a light-dependent manner, perhaps through negative modulation of the BLR proteins. This is the first evidence for a regulatory role of photolyase, a role usually attributed to cryptochromes.
The flavoprotein cryptochromes (CRYs) act as blue-light receptors in plants and insects, but perform light-independent functions at the core of the mammalian circadian clock. To drive clock oscillations, mammalian CRYs associate with the Period proteins (PERs) and together inhibit the transcription of their own genes. The SCFFbxl3 ubiquitin ligase complex controls this negative feedback loop by promoting CRY ubiquitylation and degradation. Yet, the molecular mechanisms of their interactions and the functional role of flavin adenine dinucleotide (FAD) binding in CRYs remain poorly understood. Here we report crystal structures of mammalian CRY2 in its apo, FAD-bound, and Fbxl3-Skp1-complexed forms. Distinct from other cryptochromes of known structures, mammalian CRY2 binds FAD dynamically with an open cofactor pocket. Strikingly, the F-box protein Fbxl3 captures CRY2 by simultaneously occupying its FAD-binding pocket with a conserved C-terminal tail and burying its PER-binding interface. This novel F-box protein-substrate bipartite interaction is susceptible to disruption by both FAD and PERs, suggesting a new avenue for pharmacological targeting of the complex and a multifaceted regulatory mechanism of CRY ubiquitylation.
The blue light receptors cryptochromes mediate various light responses in plants. The photoexcited cryptochrome molecules undergo a number of biophysical and biochemical changes, including electron transfer, phosphorylation, and ubiquitination, resulting in conformational changes to propagate light signals. Two modes of cryptochrome signal transduction have been recently discovered, the CIB (cryptochrome-interacting basic-helix-loop-helix 1)-dependent CRY2 regulation of transcription and the SPA1/COP1 (SUPPRESSOR OF PHYA /CONSTITUTIVELY PHOTOMORPHOGENIC1)-dependent cryptochrome regulation of proteolysis. Both cryptochrome signaling pathways rely on blue light-dependent interactions between the cryptochrome photoreceptor and its signaling proteins to modulate gene expression changes in response to blue light, leading to altered developmental programs of plants.
The cyclobutane pyrimidine dimer (CPD) and 6–4 lesion formations along with the specific breaks on strands are the most common type of DNA damage caused by Ultraviolet light (UV) irradiation. CPD photolyase I and II construct two subfamilies of flavoproteins, which have recognition and repair capabilities of CPD sites on both single stranded (ssDNA) and double stranded DNA (dsDNA) with the aid of blue light energy. The other types of flavoprotein family consist of cryptochromes (CRY) that act as photoreceptors in plants, or circadian rhythm regulators in animals. Recent findings showed that a specific type of Cryptochrome-Drosophila, Arabidopsis, Synechocystis, Human (CRY-DASH) has photorepair activity on ssDNA. In this work, real-time interactions between CRY-DASH and ss/dsDNA as well as the interactions between Vibrio cholerae photolyase (VcPHR) and ss/dsDNA were investigated using Surface Plasmon Resonance (SPR). The interactions were then characterized and compared in order to investigate the effect of different types of flavoprotein on UV damaged ss/dsDNA. SPR results confirm the specific binding of VcPHR and CRY-DASH with UV treated DNA. This study is the first instance to quantify the interactions of UV treated and untreated DNA with flavoproteins.