DNA Photolyase Research Articles

DFT/MM Simulations for Cycloreversion Reaction of Cyclobutane Pyrimidine Dimer with Deprotonated and Protonated E283.

DNA photolyase targets the primary ultraviolet (UV)-induced DNA lesion─cyclobutane pyrimidine dimer (CPD), attaches to it, and catalyzes its dissociation. The catalytic mechanism of DNA photolyase and the role of the conserved residue E283 remain subjects of debate. This study employs two-dimensional potential energy surface maps and minimum free energy paths calculated at the ωB97XD/6-31G/MM level to elucidate these mechanisms. Results suggest that the catalytic process follows a sequential, stepwise reaction in which the C5-C5 and C6-C6 bonds are cleaved in order, facilitated by a protonated E283. Activation free energies for these cleavages are calculated at 4.4 and 4.2 kcal·mol-1, respectively. Protonation of E283 reduces electrostatic repulsion with CPD and forms dual hydrogen bonds with it and provides better solvation, stabilizing the CPD radical anion, particularly during intermediate state. This stabilization renders the initial splitting step exergonic, slows reverse reactions of the C5-C5 bond cleavage and electron transfer, and ensures a high quantum yield. Furthermore, the protonation state of E283 significantly affects the type of bond cleavage. Other residues in the active site were also investigated for their roles in the mechanism.

A structural decryption of cryptochromes.

Cryptochromes (CRYs), which are signaling proteins related to DNA photolyases, play pivotal roles in sensory responses throughout biology, including growth and development, metabolic regulation, circadian rhythm entrainment and geomagnetic field sensing. This review explores the evolutionary relationships and functional diversity of cryptochromes from the perspective of their molecular structures. In general, CRY biological activities derive from their core structural architecture, which is based on a Photolyase Homology Region (PHR) and a more variable and functionally specific Cryptochrome C-terminal Extension (CCE). The α/β and α-helical domains within the PHR bind FAD, modulate redox reactive residues, accommodate antenna cofactors, recognize small molecules and provide conformationally responsive interaction surfaces for a range of partners. CCEs add structural complexity and divergence, and in doing so, influence photoreceptor reactivity and tailor function. Primary and secondary pockets within the PHR bind myriad moieties and collaborate with the CCEs to tune recognition properties and propagate chemical changes to downstream partners. For some CRYs, changes in homo and hetero-oligomerization couple to light-induced conformational changes, for others, changes in posttranslational modifications couple to cascades of protein interactions with partners and effectors. The structural exploration of cryptochromes underscores how a broad family of signaling proteins with close relationship to light-dependent enzymes achieves a wide range of activities through conservation of key structural and chemical properties upon which function-specific features are elaborated.

A circadian clock protein cryptochrome inhibits the expression of inflammatory cytokines in Chinese mitten crab (Eriocheir sinensis)

Cryptochrome (Cry), as important flavoprotein, plays a key role in regulating the innate immune response, such as the release of inflammatory cytokines. In the present study, a cryptochrome homologue (EsCry) was identified from Chinese mitten crab Eriocheir sinensis, which contained a typical DNA photolyase domain, a FAD binding domain. The transcripts of EsCry were highly expressed at 11:00, and lowest at 3:00 within one day, while those of Interleukin enhancer binding factor (EsILF), Lipopolysaccharide-induced TNF-alpha factor (EsLITAF), Tumor necrosis factor (EsTNF) and Interleukin-16 (EsIL-16) showed a rhythm expression pattern contrary to EsCry. After EsCry was knocked down by dsEsCry injection, mRNA transcripts of Timeless (EsTim), Cycle (EsCyc), Circadian locomotor output cycles kaput (EsClock), Period (EsPer), and EsLITAF, EsTNF, EsILF, EsIL-16, as well as phosphorylation level of Dorsal significantly up-regulated. The transcripts of EsLITAF, EsTNF, EsILF, and EsIL-16 in EsCry-RNAi crabs significantly down-regulated after injection of NF-κB inhibitor. The interactions of EsCyc and EsCry, EsCyc and Dorsal were observed in vitro. These results indicated that EsCry negatively regulated the expression of the cytokine TNF and IL-16 via inhibiting their transcription factor LITAF and ILF through NF-κB signaling pathway, which provide evidences to better understand the circadian regulation mechanism of cytokine production in crabs.

DNA repair and oxidative stress defense systems in radiation-resistant Deinococcus murrayi.

Deinococcus murrayi is a bacterium isolated from hot springs in Portugal, and named after Dr. Robert G.E. Murray in recognition of his research on the genus Deinococcus. Like other Deinococcus species, D. murrayi is extremely resistant to ionizing radiation. Repair of massive DNA damage and limitation of oxidative protein damage are two important factors contributing to the robustness of Deinococcus bacteria. Here, we identify, among others, the DNA repair and oxidative stress defense proteins in D. murrayi, and highlight special features of D. murrayi. For DNA repair, D. murrayi does not contain a standalone uracil-DNA glycosylase (Ung), but it encodes a protein in which Ung is fused to a DNA photolyase domain (PhrB). UvrB and UvrD contain large insertions corresponding to inteins. One of its endonuclease III enzymes lacks a [4Fe-4S] cluster. Deinococcus murrayi possesses a homolog of the error-prone DNA polymerase IV. Concerning oxidative stress defense, D. murrayi encodes a manganese catalase in addition to a heme catalase. Its organic hydroperoxide resistance protein Ohr is atypical because the redox active cysteines are present in a CXXC motif. These and other characteristics of D. murrayi show further diversity among Deinococcus bacteria with respect to resistance-associated mechanisms.

Characterization of binding constants and repair quantum yields of cyclobutane pyrimidine dimer by various class II DNA photolyase mutations

Photolyase is a flavoprotein that repairs DNA damage caused by solar ultraviolet radiation. The repair quantum yields of the class I photolyases are around 80%, and for the class II photolyases are around 50%. In this research, we designed six key residues at the active site of class II MmPL from Methanosarcina mazei to systematically characterize the binding constants and quantum yields. We used titration experiments to detect the binding affinity of MmPL wild-type and the mutants with the oligomer containing T<>T lesion. Based on the dissociation constants, we can calculate the enzyme-substrate complex concentrations and obtained the repair quantum yields. Through mutation, we unexpectedly obtained higher binding affinity and higher repair quantum yields than the wild type, suggesting the other role of some residues. These results are critical to studies of ultrafast repair dynamics to solve the repair photocycles.

A Study on Yeast Using the Photoreactivation Process to Repair the Pyrimidine Dimer Mutations

Sunlight has an indispensable importance for living things in nature [1-3]. However, the direct absorption of UV will lead to the formation of pyrimidine dimers between adjacent pyrimidines in DNA strands usually in the form of cyclobutene pyrimidine dimers (CPDs) and pyrimidine (6-4) pyrimidone photoproducts (6-4PPs) which causes great damage [4-6]. A DNA repair system, known as photoreactivation, can effectively repair the dimers using photolyase [7-9], which has currently been found in plants, prokaryotic and eukaryotic cells [10-12]. This study was carried out to determine whether photolyase DNA repair can be observed in yeast. Several yeast Petri dishes were treated with ultraviolet radiation, different treatments were then added to them, and the colonies were counted after culturing, hence verifying that yeasts can use the photoreactivation process.

An Anatomy of Fungal Eye: Fungal Photoreceptors and Signalling Mechanisms.

Organisms have developed different features to capture or sense sunlight. Vertebrates have evolved specialized organs (eyes) which contain a variety of photosensor cells that help them to see the light to aid orientation. Opsins are major photoreceptors found in the vertebrate eye. Fungi, with more than five million estimated members, represent an important clade of living organisms which have important functions for the sustainability of life on our planet. Light signalling regulates a range of developmental and metabolic processes including asexual sporulation, sexual fruit body formation, pigment and carotenoid production and even production of secondary metabolites. Fungi have adopted three groups of photoreceptors: (I) blue light receptors, White Collars, vivid, cryptochromes, blue F proteins and DNA photolyases, (II) red light sensors, phytochromes and (III) green light sensors and microbial rhodopsins. Most mechanistic data were elucidated on the roles of the White Collar Complex (WCC) and the phytochromes in the fungal kingdom. The WCC acts as both photoreceptor and transcription factor by binding to target genes, whereas the phytochrome initiates a cascade of signalling by using mitogen-activated protein kinases to elicit its cellular responses. Although the mechanism of photoreception has been studied in great detail, fungal photoreception has not been compared with vertebrate vision. Therefore, this review will mainly focus on mechanistic findings derived from two model organisms, namely Aspergillus nidulans and Neurospora crassa and comparison of some mechanisms with vertebrate vision. Our focus will be on the way light signalling is translated into changes in gene expression, which influences morphogenesis and metabolism in fungi.

Adenine Fine-Tunes DNA Photolyase's Repair Mechanism.

The comparative study of DNA repair by mesophilic and extremophilic photolyases helps us understand the evolution of these enzymes and their role in preserving life on our changing planet. The mechanism of repair of cyclobutane pyrimidine dimer lesions in DNA by electron transfer from the flavin adenine dinucleotide cofactor is the subject of intense interest. The role of adenine in mediating this process remains unresolved. Using microsecond molecular dynamics simulations, we find that adenine mediates the electron transfer in both mesophile and extremophile DNA photolyases through a similar mechanism. In fact, in all photolyases studied, the molecular conformations with the largest electronic couplings between the enzyme cofactor and DNA show the presence of adenine in 10-20% of the strongest-coupling tunneling pathways between the atoms of the electron donor and acceptor. Our theoretical analysis finds that adenine serves the critical role of fine-tuning rather than maximizing the donor-acceptor coupling within the range appropriate for the repair function.

Genomic Analysis of Haloarchaea from Diverse Environments, including Permian Halite, Reveals Diversity of Ultraviolet Radiation Survival and DNA Photolyase Gene Variants

Ultraviolet (UV) radiation responses of extremophilic and archaeal microorganisms are of interest from evolutionary, physiological, and astrobiological perspectives. Previous studies determined that the halophilic archaeon, Halobacterium sp. NRC-1, which survives in multiple extremes, is highly tolerant of UV radiation. Here, Halobacterium sp. NRC-1 UV tolerance was compared to taxonomically diverse Haloarchaea isolated from high-elevation salt flats, surface warm and cold hypersaline lakes, and subsurface Permian halite deposits. Haloterrigena/Natrinema spp. from subsurface halite deposits were the least tolerant after exposure to photoreactivating light. This finding was attributed to deviation of amino acid residues in key positions in the DNA photolyase enzyme or to the complete absence of the photolyase gene. Several Halobacterium, Halorubrum and Salarchaeum species from surface environments exposed to high solar irradiance were found to be the most UV tolerant, and Halorubrum lacusprofundi from lake sediment was of intermediate character. These results indicate that high UV tolerance is not a uniform character trait of Haloarchaea and is likely reflective of UV exposure experienced in their environment. This is the first report correlating natural UV tolerance to photolyase gene functionality among Haloarchaea and provides insights into their survival in ancient halite deposits and potentially on the surface of Mars.

Dynamic interplays between three redox cofactors in a DNA photolyase revealed by spectral decomposition

DNA repair catalyzed by photolyases is accomplished by a light-dependent electron transfer event from a fully reduced flavin adenine dinucleotide to a DNA lesion site. Prokaryotic DNA photolyase, PhrB, possesses a ribolumazine cofactor and a four-iron-four-sulfur cluster in addition to the catalytic flavin, but their functional roles are poorly understood. Here, we employ time-resolved absorption spectroscopy to probe light-induced responses in both solution and single crystals of PhrB. We jointly analyze a large collection of light-induced difference spectra from the wild-type and mutant PhrB obtained under different light and redox conditions. By applying singular value decomposition to 159 time series, we dissect light-induced spectral changes and examine the dynamic interplay between three cofactors. Our findings suggest that these cofactors form an interdependent redox network to coordinate light-induced redox responses. We propose that the ribolumazine cofactor serves as a photoprotective pigment under intense light or prolonged illumination, while the iron-sulfur cluster acts as a transient electron cache to maintain balance between two otherwise independent photoreactions of the flavin and ribolumazine.

Ultrafast repair dynamics of CPD photolesion isosteres by DNA photolyase.

UV-B rays from sunlight can cause damage to DNA by inducing photolesions, two major forms being the cyclobutane pyrimidine dimer (CPD) and a less frequently formed 6-4 pyrimidine pyrimidone (64PP) photoproduct. Photolyases (PLs) are flavoenzymes which identify these photolesions and repair them using blue light. CPD PLs utilize 10 ultrafast elementary steps, including 7 electron transfer (ET) steps, to complete the photorepair cycle and modulate the quantum repair yield1. This can be influenced by the interplay between various molecular factors at the active site: amino acid residues, the bound substrate, solvation activity and plasticity/flexibility. Here, we have utilized a combination of steady-state kinetic studies, ultrafast laser spectroscopy and molecular dynamics simulations to approach the role of chemical properties of the substrate and binding configuration. We have measured the binding constants, quantum repair yields and ultrafast reaction rates of each elementary step involved for photorepair of 3 novel CPD isosteres (CPD1 and CPD2: 5’-Thymine<>Toluene-3’ adducts2; CPD3: T<>T dimer with 5’ pyranose sugar instead of furanose) by EcPL. We have found that EcPL can repair all 3 isosteres, despite weaker binding, albeit with lower repair yields. The enzyme utilizes universally conserved pathways to repair the isosteres. The binding configuration and redox properties of the substrate may play a direct role in modulating the reaction rates of intermolecular ET and intramolecular bond splitting steps in the repair photocycle, which in turn determines their overall repair quantum yield.

Augmentation of the Photoreactivation Gene in Fremyella diplosiphon Confers UV-B Tolerance.

In spite of the enormous potential of cyanobacteria as a renewable energy source, elevated UV exposure is a major impediment to their commercial viability and productivity. Fremyella diplosiphon is a widely explored cyanobacterium with great biofuel capacity due to its high lipid content. To enhance UV stress tolerance in this species, we overexpressed the photoreactivation gene (phr A) that encodes for photolyase DNA repair enzyme in the wild type F. diplosiphon (B481-WT) by genetic transformation. Our efforts resulted in a transformant (B481-ViAnSa) with a 3808-fold increase in the phr A mRNA transcript level and enhanced growth under UV-B stress. Additionally, DNA strand breaks in the transformant were significantly lower after 12 and 16 h of UV radiation, with significantly higher dsDNA recovery in B481-ViAnSa (98.1%) compared to that in B481-WT (81.5%) at 48 h post irradiation. Photosystem II recovery time in the transformant was significantly reduced (48 h) compared to that in the wild type (72 h). Evaluation of high-value fatty acid methyl esters (FAMEs) revealed methyl palmitate, the methyl ester of hexadecenoic acid (C16:0), to be the most dominant component, accounting for 53.43% of the identified FAMEs in the transformant. Results of the study offer a promising approach to enhance UV tolerance in cyanobacteria, thus paving the way to large-scale open or closed pond cultivation for commercial biofuel production.

Cryptochrome 1 is involved in anti-bacterial immunity in Japanese flounder (Paralichthys olivaceus)

Cryptochrome is a pivotal protein of the core regulatory machinery in circadian clock and participates in various biological processes including immunity. In fish, the immunological functionality of Cryptochrome is unclear. In this study, we profiled the expression pattern of Japanese flounder cryptochrome 1 (named PoCry1) and investigated its involvement in the immune response during bacterial infection. PoCry1 is approximately 75% identical in amino acid sequence to mammalian Cryptochrome 1 and possesses the conserved DNA photolyase domain and FAD binding domain. Its transcription was detected in 10 flounder tissues and was reduced upon Vibrio harveyi challenge. The transcription of PoCry1 exhibited apparent rhythmic oscillation upon the light actuation of a day-night cycle in different tissues. The knockdown of PoCry1 resulted in significant augment of inflammatory cytokine expression. During bacterial infection, PoCry1 silencing led to increase in cytokine expression and decrease in bacterial dissemination, which could be neutralized by a specific PKA inhibitor, suggesting PoCry1 is likely able to negatively modulate the immune response to bacterial infection through PKA associated pathways.Taken together, our results revealed that PoCry1 is circadian oscillated in transcription upon rhythmic light stimulation and involved in anti-infection immunity, thus enlightening that light condition as a circadian zeitgeber could be exploited to strength fish immune system and benefit disease control in aquaculture.

Multiple Photolyases Protect the Marine Cyanobacterium Synechococcus from Ultraviolet Radiation.

ABSTRACTMarine cyanobacteria depend on light for photosynthesis, restricting their growth to the photic zone. The upper part of this layer is exposed to strong UV radiation (UVR), a DNA mutagen that can harm these microorganisms. To thrive in UVR-rich waters, marine cyanobacteria employ photoprotection strategies that are still not well defined. Among these are photolyases, light-activated enzymes that repair DNA dimers generated by UVR. Our analysis of genomes of 81 strains of Synechococcus, Cyanobium, and Prochlorococcus isolated from the world’s oceans shows that they possess up to five genes encoding different members of the photolyase/cryptochrome family, including a photolyase with a novel domain arrangement encoded by either one or two separate genes. We disrupted the putative photolyase-encoding genes in Synechococcus sp. strain RS9916 and discovered that each gene contributes to the overall capacity of this organism to survive UVR. Additionally, each conferred increased survival after UVR exposure when transformed into Escherichia coli lacking its photolyase and SOS response. Our results provide the first evidence that this large set of photolyases endows Synechococcus with UVR resistance that is far superior to that of E. coli, but that, unlike for E. coli, these photolyases provide Synechococcus with the vast majority of its UVR tolerance.

The Antarctic Moss Pohlia nutans Genome Provides Insights Into the Evolution of Bryophytes and the Adaptation to Extreme Terrestrial Habitats.

The Antarctic continent has extreme natural environment and fragile ecosystem. Mosses are one of the dominant floras in the Antarctic continent. However, their genomic features and adaptation processes to extreme environments remain poorly understood. Here, we assembled the high-quality genome sequence of the Antarctic moss (Pohlia nutans) with 698.20 Mb and 22 chromosomes. We found that the high proportion of repeat sequences and a recent whole-genome duplication (WGD) contribute to the large size genome of P. nutans when compared to other bryophytes. The genome of P. nutans harbors the signatures of massive segmental gene duplications and large expansions of gene families, likely facilitating neofunctionalization. Genomic characteristics that may support the Antarctic lifestyle of this moss comprise expanded gene families involved in phenylpropanoid biosynthesis, unsaturated fatty acid biosynthesis, and plant hormone signal transduction. Additional contributions include the significant expansion and upregulation of several genes encoding DNA photolyase, antioxidant enzymes, flavonoid biosynthesis enzymes, possibly reflecting diverse adaptive strategies. Notably, integrated multi-omic analyses elucidate flavonoid biosynthesis may function as the reactive oxygen species detoxification under UV-B radiation. Our studies provide insight into the unique features of the Antarctic moss genome and their molecular responses to extreme terrestrial environments.

Serial crystallography captures dynamic control of sequential electron and proton transfer events in a flavoenzyme.

Flavin coenzymes are universally found in biological redox reactions. DNA photolyases, with their flavin chromophore (FAD), utilize blue light for DNA repair and photoreduction. The latter process involves two single-electron transfers to FAD with an intermittent protonation step to prime the enzyme active for DNA repair. Here we use time-resolved serial femtosecond X-ray crystallography to describe how light-driven electron transfers trigger subsequent nanosecond-to-microsecond entanglement between FAD and its Asn/Arg-Asp redox sensor triad. We found that this key feature within the photolyase-cryptochrome family regulates FAD re-hybridization and protonation. After first electron transfer, the FAD•- isoalloxazine ring twists strongly when the arginine closes in to stabilize the negative charge. Subsequent breakage of the arginine-aspartate salt bridge allows proton transfer from arginine to FAD•-. Our molecular videos demonstrate how the protein environment of redox cofactors organizes multiple electron/proton transfer events in an ordered fashion, which could be applicable to other redox systems such as photosynthesis.

Reduced Molecular Flavins as Single-Electron Reductants after Photoexcitation.

Flavoenzymes mediate a multitude of chemical reactions and are catalytically active both in different oxidation states and in covalent adducts with reagents. The transfer of such reactivity to the organic laboratory using simplified molecular flavins is highly desirable, and such applications in (photo)oxidation reactions are already established. However, molecular flavins have not been used for the reduction of organic substrates yet, although this activity is known and well-studied for DNA photolyase enzymes. We report a catalytic method using reduced molecular flavins as photoreductants and γ-terpinene as a sacrificial reductant. Additionally, we present our design for air-stable, reduced flavin catalysts, which is based on a conformational bias strategy and circumvents the otherwise rapid reduction of O2 from air. Using our catalytic strategy, we were able to replace superstoichiometric amounts of the rare-earth reductant SmI2 in a 5-exo-trig cyclization of substituted barbituric acid derivatives. Such flavin-catalyzed reductions are anticipated to be beneficial for other transformations as well and their straightforward synthesis indicates future use in stereo- as well as site-selective transformations.

Identification and characterisation of blue light photoreceptor gene family and their expression in tomato (Solanum lycopersicum) under cold stress.

The Arabidopsis thaliana L. photoreceptor genes homologues in tomato (Solanum lycopersicum L.) genome were analysed using bioinformatic tools. The expression pattern of these genes under cold stress was also evaluated. Transcriptome analysis of the tomato sequence revealed that the photoreceptor gene family is involved in abiotic stress tolerance. They participate in various pathways and controlling multiple metabolic processes. They are structurally related to PAS, LIGHT-OXYGEN-VOLTAGE-SENSING (LOV), DNA photolyase, 5,10-methenyl tetrahydrofolate (MTHF), flavin-binding kelch F-box, GAF, PHY, Seven-bladed β-propeller and C27 domains. They also interact with flavin adenine dinucleotide (FAD), (5S)-5-methyl-2-(methylsulfanyl)-5-phenyl-3-(phenylamino)-3,5-dihydro-4H-imidazol-4-one (FNM) and Phytochromobilin (PϕB) ligands. These interactions help to create a cascade of protein phosphorylation involving in cell defence transcription or stress-regulated genes. They localisation of these gene families on tomato chromosomes appeared to be uneven. Phylogenetic tree of tomato and Arabidopsis photoreceptor gene family were classified into eight subgroups, indicating gene expression diversity. Morphological and physiological assessment revealed no dead plant after 4h of cold treatment. All the plants were found to be alive, but there were some variations in the data across different parameters. Cold stress significantly reduced the rate of photosynthesis from 10.06 to 3.16μmolm-2 s-1 , transpiration from 4.6 to 1.3mmolm-2 s-1 , and stomatal conductance from 94.6 to 25.6mmolm-2 s-1 . The cold stressed plants also had reduced height, root/shoot length, and fresh/dry biomass weight than the control plants. Relative expression analysis under cold stress revealed that after 4h, light stimulates the transcript level of Cry2 from 1.9 to 5.7 and PhyB from 0.98 to 6.9 compared to other photoreceptor genes.

Dynamic Interplays Between Three Redox Cofactors in DNA Photolyase PhrB

Dynamic Interplays Between Three Redox Cofactors in DNA Photolyase PhrB

Simultaneously monitoring UVC-induced DNA damage and photoenzymatic repair of cyclobutane pyrimidine dimers by electrochemical impedance spectroscopy

Cyclobutane pyrimidine dimers (CPDs) are the major DNA photoproducts of thymine-thymine dinucleotides upon ultraviolet (UV) irradiation. Failure in the repair of damaged DNA may lead to DNA replication errors, DNA mutations, and even cell death. Photoreactivation can mediate the repair of UV-induced DNA lesions by photolyases upon UVA (315–400 nm) or blue light (400–500 nm) irradiation. Herein, the UVC (254 nm)-induced DNA damage and photoenzymatic repair of the CPD products were simultaneously monitored by electrochemical impedance spectroscopy (EIS). The UVC-damaged dT20 was first immobilized on the gold electrode, and the specific recognition by the anti-CPD antibody leads to significantly increased EIS signals. The electron transfer resistance (Ret) values were linearly proportional to the concentrations of damaged dT20 ranging from 0.005 to 0.1 μM, and a detection limit of 3.06 nM was achieved. Using surface plasmon resonance, the equilibrium dissociation constant (KD) between the CPDs in dT20 and anti-CPD antibody was estimated to be (3.32 ± 0.31) × 10−12 M, indicating the strong binding affinity. Evidenced by EIS, the CPDs in the damaged dT20 could be repaired by the attached DNA photolyase under UVA (365 nm) photoexcitation, and the detachment of the photolyase from the DNA strand was accomplished after completion of the repair process. The repair efficiency was calculated to be 70.0% by EIS, being consistent with that of 71.4% by UV spectroscopy. The electrochemical method is simple, sensitive and straightforward, holding great potential for assaying other types of DNA lesions and their repair processes.