Crystal structure of cryptochrome 3 from Arabidopsis thaliana and its implications for photolyase activity.
ABSTRACT: 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: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: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: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: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 and DNA photolyases are related flavoproteins with flavin adenine dinucleotide as the common cofactor. Whereas photolyases repair DNA lesions caused by UV radiation, cryptochromes generally lack repair activity but act as UV-A/blue light photoreceptors. Two distinct electron transfer (ET) pathways have been identified in DNA photolyases. One pathway uses within its catalytic cycle, light-driven electron transfer from FADH(-)* to the DNA lesion and electron back-transfer to semireduced FADH(o) after photoproduct cleavage. This cyclic ET pathway seems to be unique for the photolyase subfamily. The second ET pathway mediates photoreduction of semireduced or fully oxidized FAD via a triad of aromatic residues that is conserved in photolyases and cryptochromes. The 5,10-methenyltetrahydrofolate (5,10-methenylTHF) antenna cofactor in members of the photolyase family is bleached upon light excitation. This process has been described as photodecomposition of 5,10-methenylTHF. We show that photobleaching of 5,10-methenylTHF in Arabidopsis cry3, a member of the cryptochrome DASH family, with repair activity for cyclobutane pyrimidine dimer lesions in single-stranded DNA and in Escherichia coli photolyase results from reduction of 5,10-methenylTHF to 5,10-methyleneTHF that requires the intact tryptophan triad. Thus, a third ET pathway exists in members of the photolyase family that remained undiscovered so far.
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: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.
Project description: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: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/
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.