The binding structure and affinity of photodamaged duplex DNA with members of the photolyase/cryptochrome family: A computational study.
ABSTRACT: 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: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 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:Homologous flavoproteins from the photolyase (PHR)/cryptochrome (CRY) family use the FAD cofactor in PHRs to catalyze DNA repair and in CRYs to tune the circadian clock and control development. To help address how PHR/CRY members achieve these diverse functions, we determined the crystallographic structure of Arabidopsis thaliana (6-4) PHR (UVR3), which is strikingly (>65%) similar in sequence to human circadian clock CRYs. The structure reveals a substrate-binding cavity specific for the UV-induced DNA lesion, (6-4) photoproduct, and cofactor binding sites different from those of bacterial PHRs and consistent with distinct mechanisms for activities and regulation. Mutational analyses were combined with this prototypic structure for the (6-4) PHR/clock CRY cluster to identify structural and functional motifs: phosphate-binding and Pro-Lys-Leu protrusion motifs constricting access to the substrate-binding cavity above FAD, sulfur loop near the external end of the Trp electron-transfer pathway, and previously undefined C-terminal helix. Our results provide a detailed, unified framework for investigations of (6-4) PHRs and the mammalian CRYs. Conservation of key residues and motifs controlling FAD access and activities suggests that regulation of FAD redox properties and radical stability is essential not only for (6-4) photoproduct DNA repair, but also for circadian clock-regulating CRY functions. The structural and functional results reported here elucidate archetypal relationships within this flavoprotein family and suggest how PHRs and CRYs use local residue and cofactor tuning, rather than larger structural modifications, to achieve their diverse functions encompassing DNA repair, plant growth and development, and circadian clock regulation.
Project description:Circadian rhythms control the temporal arrangement of molecular, physiological, and behavioral processes within an organism and also synchronize these processes with the external environment. A cell autonomous molecular oscillator, consisting of interlocking transcriptional/translational feedback loops, drives the approximately 24-hour duration of these rhythms. The cryptochrome protein (CRY) plays a central part in the negative feedback loop of the molecular clock by translocating to the nucleus and repressing CLOCK and BMAL1, two transcription factors that comprise the positive elements in this cycle. In order to gain insight into the inner workings of this feedback loop, we investigated the structure/function relationships of Xenopus laevis CRY1 (xCRY1) and xCRY2 in cultured cells. The C-terminal tails of both xCRY1 and xCRY2 are sufficient for their nuclear localization but achieve it by different mechanisms. Through the generation and characterization of xCRY/photolyase chimeras, we found that the second half of the photolyase homology region (PHR) of CRY is important for repression through facilitating interaction with BMAL1. Characterization of these functional domains in CRYs will help us to better understand the mechanism of the known roles of CRYs and to elucidate new intricacies of the molecular clock.
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:Cryptochrome (CRY) proteins play an essential role in regulating mammalian circadian rhythms. CRY is composed of a structured N-terminal domain known as the photolyase homology region (PHR), which is tethered to an intrinsically disordered C-terminal tail. The PHR domain is a critical hub for binding other circadian clock components such as CLOCK, BMAL1, PERIOD, or the ubiquitin ligases FBXL3 and FBXL21. While the isolated PHR domain is necessary and sufficient to generate circadian rhythms, removing or modifying the cryptochrome tails modulates the amplitude and/or periodicity of circadian rhythms, suggesting that they play important regulatory roles in the molecular circadian clock. In this commentary, we will discuss how recent studies of these intrinsically disordered tails are helping to establish a general and evolutionarily conserved model for CRY function, where the function of PHR domains is modulated by reversible interactions with their intrinsically disordered tails. Video abstract.
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.
Project description:Cryptochromes are evolutionarily related to the light-dependent DNA repair enzyme photolyase, serving as major regulators of circadian rhythms in insects and vertebrate animals. There are two types of cryptochromes in the animal kingdom: Drosophila-like CRYs that act as nonvisual photopigments linking circadian rhythms to the environmental light/dark cycle, and vertebrate-like CRYs that do not appear to sense light directly, but control the generation of circadian rhythms by acting as transcriptional repressors. Some animals have both types of CRYs, while others possess only one. Cryptochromes have two domains, the photolyase homology region (PHR) and an extended, intrinsically disordered C-terminus. While all animal CRYs share a high degree of sequence and structural homology in their PHR domains, the C-termini are divergent in both length and sequence identity. Recently, cryptochrome function has been shown to extend beyond its pivotal role in circadian clocks, participating in regulation of the DNA damage response, cancer progression and glucocorticoid signaling, as well as being implicated as possible magnetoreceptors. In this review, we provide a historical perspective on the discovery of animal cryptochromes, examine similarities and differences of the two types of animal cryptochromes and explore some of the divergent roles for this class of proteins.