Evolutionary History of the Photolyase/Cryptochrome Superfamily in Eukaryotes.
ABSTRACT: Photolyases and cryptochromes are evolutionarily related flavoproteins, which however perform distinct physiological functions. Photolyases (PHR) are evolutionarily ancient enzymes. They are activated by light and repair DNA damage caused by UV radiation. Although cryptochromes share structural similarity with DNA photolyases, they lack DNA repair activity. Cryptochrome (CRY) is one of the key elements of the circadian system in animals. In plants, CRY acts as a blue light receptor to entrain circadian rhythms, and mediates a variety of light responses, such as the regulation of flowering and seedling growth.We performed a comprehensive evolutionary analysis of the CRY/PHR superfamily. The superfamily consists of 7 major subfamilies: CPD class I and CPD class II photolyases, (6-4) photolyases, CRY-DASH, plant PHR2, plant CRY and animal CRY. Although the whole superfamily evolved primarily under strong purifying selection (average ? = 0.0168), some subfamilies did experience strong episodic positive selection during their evolution. Photolyases were lost in higher animals that suggests natural selection apparently became weaker in the late stage of evolutionary history. The evolutionary time estimates suggested that plant and animal CRYs evolved in the Neoproterozoic Era (~1000-541 Mya), which might be a result of adaptation to the major climate and global light regime changes occurred in that period of the Earth's geological history.
Project description:Photolyases (PHRs) and cryptochromes (CRYs) belong to the same family known as blue-light photoreceptors. Although their amino acid sequences and corresponding structures are similar to each other, they exert different functions. PHRs function as an enzyme to repair UV-induced deoxyribonucleic acid (DNA) lesions such as a cyclobutane pyrimidine dimer (CPD) and a (6-4) photoproduct ((6-4)pp), whereas CRYs are a circadian photoreceptor in plants and animals and at the same time they control the photoperiodic induction of flowering in plants. When a new type cryptochrome was identified, it was assumed that another type of CRYs, cryptochrome-DASH (CRY-DASH), which is categorized as a subfamily of photolyase/cryptochrome family, would possess the DNA photolyase activity. However, CRY-DASH had a weak DNA photolyase activity, but the reason for this is still unclear. To clarify the reason, we performed molecular dynamics (MD) simulations for a complex of CPD-PHR or CRY-DASH with damaged double-stranded DNA (dsDNA) and estimated the binding free energy, ?Gbind, between the protein and the damaged dsDNA by using a molecular mechanics/Poisson-Boltzmann surface area (MM/PBSA) method. ?Gbind for both proteins were -35 and 57 kcal mol-1, respectively, indicating that the structural stability of CRY-DASH was lower than that of CPD-PHR upon the damaged dsDNA binding. In particular, the number of amino acid residues relevant to the damaged dsDNA binding on the CRY-DASH surface was smaller than that on CPD-PHR. Therefore, the present result suggests that CRY-DASH has a weak DNA photolyase activity because it has a lower binding affinity than CPD-PHR.
Project description: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.
Project description:DNA photolyases and cryptochromes (cry) form a family of flavoproteins that use light energy in the blue/UV-A region for the repair of UV-induced DNA lesions or for signaling, respectively. Very recently, it was shown that members of the DASH cryptochrome subclade repair specifically cyclobutane pyrimidine dimers (CPDs) in UV-damaged single-stranded DNA. Here, we report the crystal structure of Arabidopsis cryptochrome 3 with an in-situ-repaired CPD substrate in single-stranded DNA. The structure shows a binding mode similar to that of conventional DNA photolyases. Furthermore, CPD lesions in double-stranded DNA are bound and repaired with similar efficiency as in single-stranded DNA if the CPD lesion is present in a loop structure. Together, these data reveal that DASH cryptochromes catalyze light-driven DNA repair like conventional photolyases but lack an efficient flipping mechanism for interaction with CPD lesions within duplex DNA.
Project description:Cryptochromes (Crys) are light sensing receptors that are present in all eukaryotes. They mainly absorb light in the UV/blue spectrum. The extant Crys consist of two subfamilies, which are descendants of photolyases but are now involved in the regulation of circadian rhythms. So far, knowledge about the evolution, phylogeny, and expression of cry genes is still scarce. The inclusion of cry sequences from a wide range of bilaterian species allowed us to analyze their phylogeny in detail, identifying six major Cry subgroups. Selective gene inactivations and stabilizations in multiple chordate as well as arthropod lineages suggest several sub- and/or neofunctionalization events. An expression study performed in zebrafish, the model organism harboring the largest amount of crys, showed indeed only partially overlapping expression of paralogous mRNA, supporting gene sub- and/or neofunctionalization. Moreover, the daily cry expression in the adult zebrafish retina indicated varying oscillation patterns in different cell types. Our extensive phylogenetic analysis provides for the first time an overview of cry evolutionary history. Although several, especially parasitic or blind species, have lost all cry genes, crustaceans have retained up to three crys, teleosts possess up to seven, and tetrapods up to four crys. The broad and cyclic expression pattern of all cry transcripts in zebrafish retinal layers implies an involvement in retinal circadian processes and supports the hypothesis of several autonomous circadian clocks present in the vertebrate retina.
Project description:Cryptochromes use near-UV/blue light to regulate a variety of growth and adaptive process. Recent biochemical studies demonstrate that the Cryptochrome-Drosophila, Arabidopsis, Synechocystis, Human (Cry-DASH) subfamily of cryptochromes have photolyase activity exclusively for single-stranded cyclobutane pyrimidine dimer (CPD)-containing DNA substrate [Selby C, Sancar A (2006) Proc Natl Acad Sci USA 103:17696-17700]. The crystal structure of cryptochrome 3 from Arabidopsis thaliana (At-Cry3), a member of the Cry-DASH proteins, at 2.1 A resolution, reveals that both the light-harvesting cofactor 5,10-methenyl-tetrahydrofolyl-polyglutamate (MTHF) and the catalytic cofactor flavin adenine dinucleotide (FAD) are noncovalently bound to the protein. The residues responsible for binding of MTHF in At-Cry3 are not conserved in Escherichia coli photolyase but are strongly conserved in the Cry-DASH subfamily of cryptochromes. The distance and orientation between MTHF and flavin adenine dinucleotide in At-Cry3 is similar to that of E. coli photolyase, in conjunction with the presence of electron transfer chain, suggesting the conservation of redox activity in At-Cry3. Two amino acid substitutions and the penetration of three charged side chains into the CPD-binding cavity in At-Cry3 alter the hydrophobic environment that is accommodating the hydrophobic sugar ring and thymine base moieties in class I CPD photolyases. These changes most likely make CPD binding less energetically favorable and, hence, insufficient to compete with pairing and stacking interactions between the CPD and the duplex DNA substrate. Thus, Cry-DASH subfamily proteins may be unable to stabilize CPD flipped out from the duplex DNA substrate but may be able to preserve the DNA repair activity toward single-stranded CPD-containing DNA substrate.
Project description: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.
Project description:DASH (Drosophila, Arabidopsis, Synechocystis, Human)-type cryptochromes (cry-DASH) belong to a family of flavoproteins acting as repair enzymes for UV-B-induced DNA lesions (photolyases) or as UV-A/blue light photoreceptors (cryptochromes). They are present in plants, bacteria, various vertebrates, and fungi and were originally considered as sensory photoreceptors because of their incapability to repair cyclobutane pyrimidine dimer (CPD) lesions in duplex DNA. However, cry-DASH can repair CPDs in single-stranded DNA, but their role in DNA repair in vivo remains to be clarified. The genome of the fungus Phycomyces blakesleeanus contains a single gene for a protein of the cryptochrome/photolyase family (CPF) encoding a cry-DASH, cryA, despite its ability to photoreactivate. Here, we show that cryA expression is induced by blue light in a Mad complex-dependent manner. Moreover, we demonstrate that CryA is capable of binding flavin (FAD) and methenyltetrahydrofolate (MTHF), fully complements the Escherichia coli photolyase mutant and repairs in vitro CPD lesions in single-stranded and double-stranded DNA with the same efficiency. These results support a role for Phycomyces cry-DASH as a photolyase and suggest a similar role for cry-DASH in mucoromycotina fungi.
Project description: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.
Project description: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.
Project description: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 for measuring light-induced difference spectra under the concentrated conditions. In the presence of a 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 not only in the cyanobacterial 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 that carry Asp at the position. We discuss potential roles for this conserved asparagine position and functional diversity in the Cry/Phr frame.