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1.  The Cryptochrome Blue Light Receptors 
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.
doi:10.1199/tab.0135
PMCID: PMC3155252  PMID: 21841916
2.  Light-Induced Conformational Changes in Full-Length Arabidopsis thaliana Cryptochrome 
Journal of molecular biology  2011;413(1):128-137.
Cryptochromes (CRYs) are widespread flavoproteins with homology to photolyases (PHRs), a class of blue-light-activated DNA repair enzymes. Unlike PHRs, both plant and animal CRYs have a C-terminal domain. This cryptochrome C-terminal (CCT) domain mediates interactions with other proteins, while the PHR-like domain converts light energy into a signal via reduction and radical formation of the flavin adenine dinucleotide cofactor. However, the mechanism by which the PHR-like domain regulates the CCT domain is not known. Here, we applied the pulsed-laser-induced transient grating method to detect conformational changes induced by blue-light excitation of full-length Arabidopsis thaliana cryptochrome 1 (AtCRY1). A significant reduction in the diffusion coefficient of AtCRY1 was observed upon photoexcitation, indicating that a large conformational change occurs in this monomeric protein. AtCRY1 containing a single mutation (W324F) that abolishes an intra-protein electron transfer cascade did not exhibit this conformational change. Moreover, the conformational change was much reduced in protein lacking the CCT domain. Thus, we conclude that the observed large conformational changes triggered by light excitation of the PHR-like domain result from C-terminal domain rearrangement. This interdomain modulation would be critical for CRYs’ ability to transduce a bluelight signal into altered protein–protein interactions for biological activity. Lastly, we demonstrate that the transient grating technique provides a powerful method for the direct observation and understanding of photoreceptor dynamics.
doi:10.1016/j.jmb.2011.08.031
PMCID: PMC4451184  PMID: 21875594
blue-light receptor; flavin; photoreduction; diffusion; conformational dynamics
3.  Cloning of the Cryptochrome-Encoding PeCRY1 Gene from Populus euphratica and Functional Analysis in Arabidopsis 
PLoS ONE  2014;9(12):e115201.
Cryptochromes are photolyase-like blue/UV-A light receptors that evolved from photolyases. In plants, cryptochromes regulate various aspects of plant growth and development. Despite of their involvement in the control of important plant traits, however, most studies on cryptochromes have focused on lower plants and herbaceous crops, and no data on cryptochrome function are available for forest trees. In this study, we isolated a cryptochrome gene, PeCRY1, from Euphrates poplar (Populus euphratica), and analyzed its structure and function in detail. The deduced PeCRY1 amino acid sequence contained a conserved N-terminal photolyase-homologous region (PHR) domain as well as a C-terminal DQXVP-acidic-STAES (DAS) domain. Secondary and tertiary structure analysis showed that PeCRY1 shares high similarity with AtCRY1 from Arabidopsis thaliana. PeCRY1 expression was upregulated at the mRNA level by light. Using heterologous expression in Arabidopsis, we showed that PeCRY1 overexpression rescued the cry1 mutant phenotype. In addition, PeCRY1 overexpression inhibited hypocotyl elongation, promoted root growth, and enhanced anthocyanin accumulation in wild-type background seedlings grown under blue light. Furthermore, we examined the interaction between PeCRY1 and AtCOP1 using a bimolecular fluorescence complementation (BiFc) assay. Our data provide evidence for the involvement of PeCRY1 in the control of photomorphogenesis in poplar.
doi:10.1371/journal.pone.0115201
PMCID: PMC4264880  PMID: 25503486
4.  Human and Drosophila Cryptochromes Are Light Activated by Flavin Photoreduction in Living Cells  
PLoS Biology  2008;6(7):e160.
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.
Author Summary
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.
doi:10.1371/journal.pbio.0060160
PMCID: PMC2443192  PMID: 18597555
5.  The cryptochromes 
Genome Biology  2005;6(5):220.
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.
doi:10.1186/gb-2005-6-5-220
PMCID: PMC1175950  PMID: 15892880
6.  The action mechanisms of plant cryptochromes 
Trends in Plant Science  2011;16(12):684-691.
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.
doi:10.1016/j.tplants.2011.09.002
PMCID: PMC3277817  PMID: 21983106
7.  A Study of the Blue-Light-Dependent Phosphorylation, Degradation, and Photobody Formation of Arabidopsis CRY2 
Molecular Plant  2012;5(3):200-207.
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.
doi:10.1093/mp/sss007
PMCID: PMC3355346  PMID: 22311776
protein phosphorylation; signal transduction; fluorescence imaging; protein degradation; photobody
8.  A Photolyase-Like Protein from Agrobacterium tumefaciens with an Iron-Sulfur Cluster 
PLoS ONE  2011;6(10):e26775.
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.
doi:10.1371/journal.pone.0026775
PMCID: PMC3204975  PMID: 22066008
9.  Genetic Analysis of Circadian Responses to Low Frequency Electromagnetic Fields in Drosophila melanogaster 
PLoS Genetics  2014;10(12):e1004804.
The blue-light sensitive photoreceptor cryptochrome (CRY) may act as a magneto-receptor through formation of radical pairs involving a triad of tryptophans. Previous genetic analyses of behavioral responses of Drosophila to electromagnetic fields using conditioning, circadian and geotaxis assays have lent some support to the radical pair model (RPM). Here, we describe a new method that generates consistent and reliable circadian responses to electromagnetic fields that differ substantially from those already reported. We used the Schuderer apparatus to isolate Drosophila from local environmental variables, and observe extremely low frequency (3 to 50 Hz) field-induced changes in two locomotor phenotypes, circadian period and activity levels. These field-induced phenotypes are CRY- and blue-light dependent, and are correlated with enhanced CRY stability. Mutational analysis of the terminal tryptophan of the triad hypothesised to be indispensable to the electron transfer required by the RPM reveals that this residue is not necessary for field responses. We observe that deletion of the CRY C-terminus dramatically attenuates the EMF-induced period changes, whereas the N-terminus underlies the hyperactivity. Most strikingly, an isolated CRY C-terminus that does not encode the Tryptophan triad nor the FAD binding domain is nevertheless able to mediate a modest EMF-induced period change. Finally, we observe that hCRY2, but not hCRY1, transformants can detect EMFs, suggesting that hCRY2 is blue light-responsive. In contrast, when we examined circadian molecular cycles in wild-type mouse suprachiasmatic nuclei slices under blue light, there was no field effect. Our results are therefore not consistent with the classical Trp triad-mediated RPM and suggest that CRYs act as blue-light/EMF sensors depending on trans-acting factors that are present in particular cellular environments.
Author Summary
Low frequency electromagnetic fields (EMFs) are associated with electrical power lines and have been implicated in the development of childhood leukemias. However, the Earth also has a natural EMF that animals can detect and which they use in order to navigate and orient themselves, particularly during migrations. One way they might do this is by using specialised photoreceptors called cryptochromes, which when activated by light, generate changes within the molecule that are susceptible to EMFs. Cryptochromes are important components of animal circadian clocks, the 24 hour timers that determine daily behavioral and physiological cycles. We have studied the circadian behavior of the fruitfly and have observed some novel and robust effects of EMFs on the fly's sleep-wake cycle that are mediated by cryptochrome. By using cryptochrome mutants we find that our results do not support the classic model for how this molecule might respond to EMFs. We also show that mammalian cryptochromes can respond to EMF when placed into transgenic Drosophila, whereas in mammalian clock neurons, they cannot. Consequently, the EMF responsiveness of cryptochrome is determined by its intracellular environment, suggesting that other, unknown molecules that interact with cryptochrome are also very important.
doi:10.1371/journal.pgen.1004804
PMCID: PMC4256086  PMID: 25473952
10.  Hypersensitive to Red and Blue 1 and Its Modification by Protein Phosphatase 7 Are Implicated in the Control of Arabidopsis Stomatal Aperture 
PLoS Genetics  2012;8(5):e1002674.
The stomatal pores are located on the plant leaf epidermis and regulate CO2 uptake for photosynthesis and the loss of water by transpiration. Their stomatal aperture therefore affects photosynthesis, water use efficiency, and agricultural crop yields. Blue light, one of the environmental signals that regulates the plant stomatal aperture, is perceived by the blue/UV-A light-absorbing cryptochromes and phototropins. The signal transduction cascades that link the perception of light to the stomatal opening response are still largely unknown. Here, we report two new players, Hypersensitive to Red and Blue 1 (HRB1) and Protein Phosphatase 7 (PP7), and their genetic and biochemical interactions in the control of stomatal aperture. Mutations in either HRB1 or PP7 lead to the misregulation of the stomatal aperture and reduce water loss under blue light. Both HRB1 and PP7 are expressed in the guard cells in response to a light-to-dark or dark-to-light transition. HRB1 interacts with PP7 through its N-terminal ZZ-type zinc finger motif and requires a functional PP7 for its stomatal opening response. HRB1 is phosphorylated in vivo, and PP7 can dephosphorylate HRB1. HRB1 is mostly dephosphorylated in a protein complex of 193 kDa in the dark, and blue light increases complex size to 285 kDa. In the pp7 mutant, this size shift is impaired, and HRB1 is predominately phosphorylated. We propose that a modification of HRB1 by PP7 under blue light is essential to acquire a proper conformation or to bring in new components for the assembly of a functional HRB1 protein complex. Guard cells control stomatal opening in response to multiple environmental or biotic stimuli. This study may furnish strategies that allow plants to enjoy the advantages of both constitutive and ABA-induced protection under water-limiting conditions.
Author Summary
Stomatal aperture is regulated by many environmental and biotic cues such as blue light, drought, elevated CO2 concentrations, high humidity, and pathogenic elicitors. Stomatal apertures vary over diurnal cycles, and stomata tend to be open during the day in response to blue light and tend to be closed at night. The blue/UV-A light-absorbing cryptochromes and phototropins are the receptors for the blue light response. We report the action of HRB1, a nuclear ZZ-type zinc finger protein, and PP7, a positive regulator of blue light signaling in the nucleus, in the signal transduction cascades downstream of blue light perception. Both hrb1 and pp7 mutants are more resistant to dehydration and show reductions in both water loss and blue light-regulated stomatal aperture. Our studies on their genetic and biochemical interactions offer novel insights on the network structure of the light signaling machinery and plant interactions with the environment. Periodic drought is one of the major environmental factors that limits biomass production and crop yield in a changing global climate. Our studies may open new possibilities to engineer plants to survive desiccation.
doi:10.1371/journal.pgen.1002674
PMCID: PMC3349726  PMID: 22589732
11.  dbCRY: a Web-based comparative and evolutionary genomics platform for blue-light receptors 
Cryptochromes are flavoproteins that play a central role in the circadian oscillations of all living organisms except archaea. Cryptochromes are clustered into three subfamilies: plant-type cryptochromes, animal-type cryptochromes and cryptochrome-DASH proteins. These subfamilies are composed of photolyase/cryptochrome superfamily with 6–4 photolyase and cyclobutane pyrimidine dimer photolyase. Cryptochromes have conserved domain architectures with two distinct domains, an N-terminal photolyase-related domain and a C-terminal domain. Although the molecular function and domain architecture of cryptochromes are conserved, their molecular mechanisms differ between plants and animals. Thus, cryptochromes are one of the best candidates for comparative and evolutionary studies. Here, we have developed a Web-based platform for comparative and evolutionary studies of cryptochromes, dbCRY (http://www.dbcryptochrome.org/). A pipeline built upon the consensus domain profile was applied to 1438 genomes and identified 1309 genes. To support comparative and evolutionary genomics studies, the Web interface provides diverse functions such as (i) browsing by species, (ii) protein domain analysis, (iii) multiple sequence alignment, (iv) homology search and (v) extended analysis opportunities through the implementation of ‘Favorite Browser’ powered by the Comparative Fungal Genomics Platform 2.0 (CFGP 2.0; http://cfgp.snu.ac.kr/). dbCRY would serve as a standardized and systematic solution for cryptochrome genomics studies.
Database URL: http://www.dbcryptochrome.org/
doi:10.1093/database/bau037
PMCID: PMC4016680  PMID: 24816342
12.  Analysis of Autophosphorylating Kinase Activities Of Arabidopsis and Human Cryptochromes ζ 
Biochemistry  2006;45(44):13369-13374.
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.
doi:10.1021/bi061556n
PMCID: PMC2527022  PMID: 17073458
13.  Crystallization and preliminary X-ray analysis of cryptochrome 3 from Arabidopsis thaliana  
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.
doi:10.1107/S1744309105028897
PMCID: PMC1991327  PMID: 16511200
cryptochrome 3; light receptors
14.  The Second Chromophore in Drosophila Photolyase/Cryptochrome Family Photoreceptors† 
Biochemistry  2011;51(1):167-171.
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.
doi:10.1021/bi201536w
PMCID: PMC3302575  PMID: 22175817
15.  Key Dynamics of Conserved Asparagine in a Cryptochrome/Photolyase Family Protein by FTIR Spectroscopy† 
Biochemistry  2010;49(41):8882-8891.
Cryptochromes (Crys) and photolyases (Phrs) are flavoproteins that contain an identical cofactor (flavin adenine dinucleotide, FAD) within the same protein architecture, but whose physiological functions are entirely different. In this study, we investigated light-induced conformational changes of a cyanobacterium Cry/Phr-like protein (SCry-DASH) with UV–visible and Fourier transform infrared (FTIR) spectroscopy. We developed a system to measure light-induced difference spectra under the concentrated conditions. In the presence of reducing agent, SCry-DASH showed photoreduction to the reduced form, and we identified a signal unique for an anionic form in the process. Difference FTIR spectra enabled us to assign characteristic FTIR bands to the respective redox forms of FAD. An asparagine residue, which anchors the FAD embedded within the protein is conserved in not only the cyanobaterial protein, but also in Phrs and other Crys including the mammalian clock-related Crys. By characterizing an asparagine-to-cysteine (N392C) mutant of SCry-DASH, which mimics an insect specific Cry, we identified structural changes of the carbonyl group of this conserved asparagine upon light-irradiation. We also found that the N392C mutant is stabilized in the anionic form. We did not observe a signal from protonated carboxylic acid residues during the reduction process, suggesting that the carboxylic acid moiety would not be directly involved as a proton donor to FAD in the system. These results are in contrast to plant specific Crys represented by Arabidopsis thaliana Cry1 which carry Asp at the position. We discuss potential roles for this conserved asparagine position and functional diversity in the Cry/Phr frame.
doi:10.1021/bi1009979
PMCID: PMC4329311  PMID: 20828134
16.  Purification and Characterization of a Type III Photolyase from Caulobacter crescentus 
Biochemistry  2008;47(39):10255-10261.
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.
doi:10.1021/bi801085a
PMCID: PMC2701293  PMID: 18771290
17.  The Potorous CPD Photolyase Rescues a Cryptochrome-Deficient Mammalian Circadian Clock 
PLoS ONE  2011;6(8):e23447.
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.
doi:10.1371/journal.pone.0023447
PMCID: PMC3156801  PMID: 21858120
18.  Investigation of Real-Time Photorepair Activity on DNA via Surface Plasmon Resonance 
PLoS ONE  2012;7(8):e44392.
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.
doi:10.1371/journal.pone.0044392
PMCID: PMC3430658  PMID: 22952969
19.  Structure of Full-length Drosophila Cryptochrome 
Nature  2011;480(7377):396-399.
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.
doi:10.1038/nature10618
PMCID: PMC3240699  PMID: 22080955
20.  Trichoderma atroviride PHR1, a Fungal Photolyase Responsible for DNA Repair, Autoregulates Its Own Photoinduction▿  
Eukaryotic Cell  2007;6(9):1682-1692.
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.
doi:10.1128/EC.00208-06
PMCID: PMC2043357  PMID: 17545314
21.  The Trichoderma reesei Cry1 Protein Is a Member of the Cryptochrome/Photolyase Family with 6–4 Photoproduct Repair Activity 
PLoS ONE  2014;9(6):e100625.
DNA-photolyases use UV-visible light to repair DNA damage caused by UV radiation. The two major types of DNA damage are cyclobutane pyrimidine dimers (CPD) and 6–4 photoproducts (6-4PP), which are repaired under illumination by CPD and 6–4 photolyases, respectively. Cryptochromes are proteins related to DNA photolyases with strongly reduced or lost DNA repair activity, and have been shown to function as blue-light photoreceptors and to play important roles in circadian rhythms in plants and animals. Both photolyases and cryptochromes belong to the cryptochrome/photolyase family, and are widely distributed in all organisms. Here we describe the characterization of cry1, a member of the cryptochrome/photolyase protein family of the filamentous fungus Trichoderma reesei. We determined that cry1 transcript accumulates when the fungus is exposed to light, and that such accumulation depends on the photoreceptor Blr1 and is modulated by Envoy. Conidia of cry1 mutants show decreased photorepair capacity of DNA damage caused by UV light. In contrast, strains over-expressing Cry1 show increased repair, as compared to the parental strain even in the dark. These observations suggest that Cry1 may be stimulating other systems involved in DNA repair, such as the nucleotide excision repair system. We show that Cry1, heterologously expressed and purified from E. coli, is capable of binding to undamaged and 6-4PP damaged DNA. Photorepair assays in vitro clearly show that Cry1 repairs 6-4PP, but not CPD and Dewar DNA lesions.
doi:10.1371/journal.pone.0100625
PMCID: PMC4070973  PMID: 24964051
22.  Functional Evolution of the Photolyase/Cryptochrome Protein Family: Importance of the C Terminus of Mammalian CRY1 for Circadian Core Oscillator Performance†  
Molecular and Cellular Biology  2006;26(5):1743-1753.
Cryptochromes (CRYs) are composed of a core domain with structural similarity to photolyase and a distinguishing C-terminal extension. While plant and fly CRYs act as circadian photoreceptors, using the C terminus for light signaling, mammalian CRY1 and CRY2 are integral components of the circadian oscillator. However, the function of their C terminus remains to be resolved. Here, we show that the C-terminal extension of mCRY1 harbors a nuclear localization signal and a putative coiled-coil domain that drive nuclear localization via two independent mechanisms and shift the equilibrium of shuttling mammalian CRY1 (mCRY1)/mammalian PER2 (mPER2) complexes towards the nucleus. Importantly, deletion of the complete C terminus prevents mCRY1 from repressing CLOCK/BMAL1-mediated transcription, whereas a plant photolyase gains this key clock function upon fusion to the last 100 amino acids of the mCRY1 core and its C terminus. Thus, the acquirement of different (species-specific) C termini during evolution not only functionally separated cryptochromes from photolyase but also caused diversity within the cryptochrome family.
doi:10.1128/MCB.26.5.1743-1753.2006
PMCID: PMC1430250  PMID: 16478995
23.  DNA damage shifts circadian clock time via Hausp-dependent Cry1 stabilization 
eLife  null;4:e04883.
The circadian transcriptional repressors cryptochrome 1 (Cry1) and 2 (Cry2) evolved from photolyases, bacterial light-activated DNA repair enzymes. In this study, we report that while they have lost DNA repair activity, Cry1/2 adapted to protect genomic integrity by responding to DNA damage through posttranslational modification and coordinating the downstream transcriptional response. We demonstrate that genotoxic stress stimulates Cry1 phosphorylation and its deubiquitination by Herpes virus associated ubiquitin-specific protease (Hausp, a.k.a Usp7), stabilizing Cry1 and shifting circadian clock time. DNA damage also increases Cry2 interaction with Fbxl3, destabilizing Cry2. Thus, genotoxic stress increases the Cry1/Cry2 ratio, suggesting distinct functions for Cry1 and Cry2 following DNA damage. Indeed, the transcriptional response to genotoxic stress is enhanced in Cry1−/− and blunted in Cry2−/− cells. Furthermore, Cry2−/− cells accumulate damaged DNA. These results suggest that Cry1 and Cry2, which evolved from DNA repair enzymes, protect genomic integrity via coordinated transcriptional regulation.
DOI: http://dx.doi.org/10.7554/eLife.04883.001
eLife digest
Many aspects of our physiology and behavior, most notably our patterns of sleep and wakefulness, are synchronized with the day–night cycle. These circadian rhythms are generated and maintained by the circadian clock, which consists of positive and negative feedback loops formed by a large number of genes and proteins. The end result is that the rates at which thousands of proteins are produced varies rhythmically over the course of the day–night cycle.
It has long been suspected that one of the functions of this circadian clock is to control the timing of cell division. Moreover, since UV radiation can give rise to genetic mutations when cells divide, it is thought that the circadian clock limits the amount of DNA damage that occurs during daytime. Papp, Huber et al. have now confirmed that the circadian clock does indeed participate in the DNA damage response and have revealed that two proteins known to be involved in the circadian clock—Cryptochrome 1 and 2—have a central role in protecting the integrity of the genetic information in the cell. These proteins evolved from light-activated enzymes that repair DNA in bacteria.
While mammalian cryptochromes have lost their ability to repair DNA, they still prefer to bind to genetic material that has been damaged by UV radiation. Papp, Huber et al. show that DNA damage triggers cryptochrome 1 to bind to a protein called Hausp, which stabilizes the cryptochrome and prevents it from being broken down. By contrast, DNA damage triggers cryptochrome 2 to bind to a protein called Fbxl3, which has a destabilizing effect on the cryptochrome and promotes its degradation. Since the cryptochromes regulate the activity of BMAL1 and CLOCK, the proteins associated with the two master clock genes, these changes can have a significant effect on the circadian clock of an organism.
Further experiments are needed to work out how these proteins influence the activity of BMAL1 and CLOCK, and to investigate the seemingly conflicting roles of the two cryptochromes and the interactions between them.
DOI: http://dx.doi.org/10.7554/eLife.04883.002
doi:10.7554/eLife.04883
PMCID: PMC4352707  PMID: 25756610
circadian rhythm; cryptochrome; Hausp; DNA damage; ubiquitin; phosphorylation; mouse
24.  Blue Light-Dependent Interaction of CRY2 with SPA1 Regulates COP1 activity and Floral Initiation in Arabidopsis 
Current biology : CB  2011;21(10):841-847.
Summary
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.
doi:10.1016/j.cub.2011.03.048
PMCID: PMC3150455  PMID: 21514160
25.  Light-induced conformational change and product release in DNA repair by (6-4) photolyase 
Proteins of the cryptochrome/photolyase family share high sequence similarities, common folds and the flavin adenine dinucleotide (FAD) cofactor, but exhibit diverse physiological functions. Mammalian cryptochromes are essential regulatory components of the 24-hour circadian clock, whereas (6-4) photolyases recognize and repair UV-induced DNA damage by using light energy absorbed by FAD. Despite increasing knowledge about physiological functions from genetic analyses, the molecular mechanisms and conformational dynamics involved in clock signaling and DNA repair remain poorly understood. The (6-4) photolyase, which has strikingly high similarity to human clock cryptochromes, is a prototypic biological system to study conformational dynamics of cryptochrome/photolyase family proteins. The entire light-dependent DNA repair process for (6-4) photolyase can be reproduced in a simple in vitro system. To decipher pivotal reactions of the common FAD cofactor, we accomplished time resolved measurements of radical formation, diffusion, and protein conformational changes during light-dependent repair by full-length (6-4) photolyase on DNA carrying a single UV-induced damage. The (6-4) photolyase by itself showed significant volume changes after blue light activation, indicating protein conformational changes distant from the flavin cofactor. A drastic diffusion change was observed only in the presence of both (6-4) photolyase and damaged DNA, and not for (6-4) photolyase alone or with undamaged DNA. Thus, we propose that this diffusion change reflects the rapid (50 μs time constant) dissociation of the protein from the repaired DNA product. Conformational changes with such fast turnover would likely enable DNA repair photolyases to access the entire genome in cells.
doi:10.1021/ja107691w
PMCID: PMC4342989  PMID: 21271694
blue light receptor; DNA damage and repair; flavoprotein; flavin adenine dinucleotide; transient grating

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