Dynamic responses of Fusarium mangiferae to ultra-violet radiation.

The repair capacity of ultra-violet (UV) light DNA damage is important for adaptation of fungi to different ecological niches. We previously showed that in the soil-borne pathogen Fusarium oxysporum photo-reactivation dependent UV repair is induced at the germling stage and reduced at the filament stage. Here, we tested the developmental control of the transcription of photolyase, UV survival, UV repair capacity, and UV induced mutagenesis in the foliar pathogen Fusarium mangiferae. Unlike F. oxysporum, neither did we observe developmental control over photo-reactivation dependent repair nor the changes in gene expression of photolyase throughout the experiment. Similarly, photo-reactivation assisted reduction in UV induced mutagenesis was similar throughout the development of F. mangiferae but fluctuated during the development of F. oxysporum. To generate hypotheses regarding the recovery of F. mangiferae after UV exposure, an RNAseq analysis was performed after irradiation at different timepoints. The most striking effect of UV on F. mangiferae was developmental-dependent induction of translation related genes. We further report a complex response that changes during recovery time and involves translation, cell cycle and lipid biology related genes.

A recombinant fungal photolyase autonomously enters human cell nuclei to fix UV-induced DNA lesions.

Solar ultraviolet radiations induced DNA damages in human skin cells with cyclobutane pyrimidine dimers (CPD) and (6-4) photoproducts (6-4PPs) as the most frequent lesions. CPDs are repaired much slower than 6-4PPs by the nucleotide excision repair pathway, which are thus the major lesions that interfere with key cellular processes and give rise to gene mutations, possibly resulting in skin cancer. In prokaryotes and multicellular eukaryotes other than placental mammals, CPDs can be rapidly repaired by CPD photolyases in one simple enzymatic reaction using the energy of blue light. In this study, we aim to construct recombinant CPD photolyases that can autonomously enter human cell nuclei to fix UV-induced CPDs. A fly cell penetration peptide and a viral nucleus localization signal peptide were recombined with a fungal CPD photolyase to construct a recombinant protein. This engineered CPD photolyase autonomously crosses cytoplasm and nuclear membrane of human cell nuclei, which then efficiently photo-repairs UV-induced CPD lesions in the genomic DNA. This further protects the cells by increasing SOD activity, and decreasing cellular ROSs, malondialdehyde and apoptosis.

Recombinant production of a highly efficient photolyase from Thermus thermophilus.

Ultraviolet (UV) radiation from sunlight can damage DNA, inducing mutagenesis and eventually leading to skin cancer. Topical sunscreens are used to avoid the effect of UV irradiation, but the topical application of DNA repair enzymes, such as photolyase, can provide active photoprotection by DNA recovery. Here we produced a recombinant Thermus thermophilus photolyase expressed in Escherichia coli, evaluated the kinetic parameters of bacterial growth and the kinetics and stability of the enzyme. The maximum biomass (𝑋𝑚𝑎𝑥 ) of 2.0 g L-1 was reached after 5 h of cultivation, corresponding to 𝑃X  = 0.4 g L-1 h. The µð‘šð‘Žð‘¥ corresponded to 1.0 h-1 . Photolyase was purified by affinity chromatography and high amounts of pure enzyme were obtained (3.25 mg L-1 of cultivation). Two different methods demonstrated the enzyme activity on DNA samples and very low enzyme concentrations, such as 15 µg mL-1 , already resulted in 90% of CPD photodamage removal. We also determined photolyase kM of 9.5 nM, confirming the potential of the enzyme at very low concentrations, and demonstrated conservation of enzyme activity after freezing (-20°C) and lyophilization. Therefore, we demonstrate T. thermophilus photolyase capacity of CPD damage repair and its potential as an active ingredient to be incorporated in dermatological products.

Poaceae plants transfer cyclobutane pyrimidine dimer photolyase to chloroplasts for ultraviolet-B resistance.

Photoreactivation enzyme that repairs cyclobutane pyrimidine dimer (CPD) induced by ultraviolet-B radiation, commonly called CPD photolyase (PHR) is essential for plants living under sunlight. Rice (Oryza sativa) PHR (OsPHR) is a unique triple-targeting protein. The signal sequences required for its translocation to the nucleus or mitochondria are located in the C-terminal region but have yet to be identified for chloroplasts. Here, we identified sequences located in the N-terminal region, including the serine-phosphorylation site at position 7 of OsPHR, and found that OsPHR is transported/localized to chloroplasts via a vesicle transport system under the control of serine-phosphorylation. However, the sequence identified in this study is only conserved in some Poaceae species, and in many other plants, PHR is not localized to the chloroplasts. Therefore, we reasoned that Poaceae species need the ability to repair CPD in the chloroplast genome to survive under sunlight and have uniquely acquired this mechanism for PHR chloroplast translocation.

Directed ultrafast conformational changes accompany electron transfer in a photolyase as resolved by serial crystallography.

Charge-transfer reactions in proteins are important for life, such as in photolyases which repair DNA, but the role of structural dynamics remains unclear. Here, using femtosecond X-ray crystallography, we report the structural changes that take place while electrons transfer along a chain of four conserved tryptophans in the Drosophila melanogaster (6-4) photolyase. At femto- and picosecond delays, photoreduction of the flavin by the first tryptophan causes directed structural responses at a key asparagine, at a conserved salt bridge, and by rearrangements of nearby water molecules. We detect charge-induced structural changes close to the second tryptophan from 1 ps to 20 ps, identifying a nearby methionine as an active participant in the redox chain, and from 20 ps around the fourth tryptophan. The photolyase undergoes highly directed and carefully timed adaptations of its structure. This questions the validity of the linear solvent response approximation in Marcus theory and indicates that evolution has optimized fast protein fluctuations for optimal charge transfer.

Cold-induced skin darkening does not protect amphibian larvae from UV-associated DNA damage.

Amphibian declines are sometimes correlated with increasing levels of ultraviolet radiation (UVR). While disease is often implicated in declines, environmental factors such as temperature and UVR play an important role in disease epidemiology. The mutagenic effects of UVR exposure on amphibians are worse at low temperatures. Amphibians from cold environments may be more susceptible to increasing UVR. However, larvae of some species demonstrate cold acclimation, reducing UV-induced DNA damage at low temperatures. Understanding of the mechanisms underpinning this response is lacking. We reared Limnodynastes peronii larvae in cool (15°C) or warm (25°C) waters before acutely exposing them to 1.5 h of high intensity (80 µW cm-2 ) UVBR. We measured the color of larvae and mRNA levels of a DNA repair enzyme. We reared larvae at 25°C in black or white containers to elicit a skin color response, and then measured DNA damage levels in the skin and remaining carcass following UVBR exposure. Cold-acclimated larvae were darker and displayed lower levels of DNA damage than warm-acclimated larvae. There was no difference in CPD-photolyase mRNA levels between cold- and warm-acclimated larvae. Skin darkening in larvae did not reduce their accumulation of DNA damage following UVR exposure. Our results showed that skin darkening does not explain cold-induced reductions in UV-associated DNA damage in L. peronii larvae. Beneficial cold-acclimation is more likely underpinned by increased CPD-photolyase abundance and/or increased photolyase activity at low temperatures.

Metarhizium pingshaense photolyase expression and virulence to Rhipicephalus microplus after UV-B exposure.

The current study examined the impact of ultraviolet (UV)-B radiation in Metarhizium pingshaense blastospores' photolyase expression and their virulence against Rhipicephalus microplus. Blastospores were exposed to UV under laboratory and field conditions. Ticks were treated topically with fungal suspension and exposed to UV-B in the laboratory for three consecutive days. The expression of cyclobutane pyrimidine dimmers (CPDs)-photolyase gene maphr1-2 in blastospores after UV exposure followed by white light exposure was accessed after 0, 8, 12, 24, 36, and 48 h. Average relative germination of blastospores 24 h after in vitro UV exposure was 8.4% lower than 48 h. Despite this, the relative germination of blastospores exposed to UV in the field 18 h (95.7 ± 0.3%) and 28 h (97.3 ± 0.8%) after exposure were not different (p > 0.05). Ticks treated with fungus and not exposed to UV exhibited 0% survival 10 days after the treatment, while fungus-treated ticks exposed to UV exhibited 50 ± 11.2% survival. Expression levels of maphr1-2 8, 12, and 24 h after UV-B exposure were not different from time zero. Maphr1-2 expression peak in M. pingshaense blastospores occurred 36 h after UV-B exposure, in the proposed conditions and times analyzed, suggesting repair mechanisms other than CPD-mediated-photoreactivation might be leading blastospores' germination from 0 to 24 h.

One More for Light-triggered Conformational Changes in Cryptochromes: CryP from Phaeodactylum tricornutum.

Cryptochromes are a ubiquitously occurring class of photoreceptors. Together with photolyases, they form the Photolyase Cryptochrome Superfamily (PCSf) by sharing a common protein architecture and binding mode of the FAD chromophore. Despite these similarities, PCSf members exert different functions. Photolyases repair UV-induced DNA damage by photocatalytically driven electron transfer between FADH¯ and the DNA lesion, whereas cryptochromes are light-dependent signaling molecules and trigger various biological processes by photoconversion of their FAD redox and charge states. Given that most cryptochromes possess a C-terminal extension (CTE) of varying length, the functions of their CTE have not yet been fully elucidated and are hence highly debated. In this study, the role of the CTE was investigated for a novel subclass of the PCSf, the CryP-like cryptochromes, by hydrogen/deuterium exchange and mass-spectrometric analysis. Striking differences in the relative deuterium uptake were observed in different redox states of CryP from the diatom Phaeodactylum tricornutum. Based on these measurements we propose a model for light-triggered conformational changes in CryP-like cryptochromes that differs from other known cryptochrome families like the insect or plant cryptochromes.

Rad2, Rad14 and Rad26 recover Metarhizium robertsii from solar UV damage through photoreactivation in vivo.

Rad2, Rad14 and Rad26 recover ultraviolet (UV) damage by nucleotide excision repair (NER) in budding yeast but their functions in filamentous fungi have not been elucidated. Here, we report mechanistically different anti-UV effects of nucleus-specific Rad2, Rad14 and Rad26 orthologs in Metarhizium robertsii, an insect-pathogenic fungus. The null mutants of rad2, rad14 and rad26 showed a decrease of ∼90% in conidial resistance to UVB irradiation. When conidia were impaired at a UVB dose of 0.15 J/cm2, they were photoreactivated (germinated) by only 6-13% through a 5-h light plus 19-h dark incubation, whereas 100%, 80% and 70% of the wild-type conidia were photoreactivated at 0.15, 0.3 and 0.4 J/cm2, respectively. The dose-dependent photoreactivation rates were far greater than the corresponding 24-h dark reactivation rates and were largely enhanced by the overexpression (OE) of rad2, rad14 or rad26 in the wild-type strain. The OE strains exhibited markedly greater activities in photoreactivation of conidia inactivated at 0.5-0.7 J/cm2 than did the wild-type strain. Confirmed interactions of Rad2, Rad14 and Rad26 with photolyase regulators and/or Rad1 or Rad10 suggest that each of these proteins could have evolved into a component of the photolyase regulator-cored protein complex to mediate photoreactivation. The interactions inhibited in the null mutants resulted in transcriptional abolishment or repression of those factors involved in the complex. In conclusion, the anti-UV effects of Rad2, Rad14 and Rad26 depend primarily on DNA photorepair-dependent photoreactivation in M. robertsii and mechanistically differ from those of yeast orthologs depending on NER.

Visualizing the DNA repair process by a photolyase at atomic resolution.

Photolyases, a ubiquitous class of flavoproteins, use blue light to repair DNA photolesions. In this work, we determined the structural mechanism of the photolyase-catalyzed repair of a cyclobutane pyrimidine dimer (CPD) lesion using time-resolved serial femtosecond crystallography (TR-SFX). We obtained 18 snapshots that show time-dependent changes in four reaction loci. We used these results to create a movie that depicts the repair of CPD lesions in the picosecond-to-nanosecond range, followed by the recovery of the enzymatic moieties involved in catalysis, completing the formation of the fully reduced enzyme-product complex at 500 nanoseconds. Finally, back-flip intermediates of the thymine bases to reanneal the DNA were captured at 25 to 200 microseconds. Our data cover the complete molecular mechanism of a photolyase and, importantly, its chemistry and enzymatic catalysis at work across a wide timescale and at atomic resolution.

Time-resolved crystallography captures light-driven DNA repair.

Photolyase is an enzyme that uses light to catalyze DNA repair. To capture the reaction intermediates involved in the enzyme's catalytic cycle, we conducted a time-resolved crystallography experiment. We found that photolyase traps the excited state of the active cofactor, flavin adenine dinucleotide (FAD), in a highly bent geometry. This excited state performs electron transfer to damaged DNA, inducing repair. We show that the repair reaction, which involves the lysis of two covalent bonds, occurs through a single-bond intermediate. The transformation of the substrate into product crowds the active site and disrupts hydrogen bonds with the enzyme, resulting in stepwise product release, with the 3' thymine ejected first, followed by the 5' base.

Filming DNA repair at the atomic level.

Dissection of multistep catalysis by a photoenzyme could inspire green chemistry applications.

Could violet-blue lights increase the bacteria resistance against ultraviolet radiation mediated by photolyases?

Studies have demonstrated bacterial inactivation by radiations at wavelengths between 400 and 500 nm emitted by low-power light sources. The phototoxic activity of these radiations could occur by oxidative damage in DNA and membrane proteins/lipids. However, some cellular mechanisms can reverse these damages in DNA, allowing the maintenance of genetic stability. Photoreactivation is among such mechanisms able to repair DNA damages induced by ultraviolet radiation, ranging from ultraviolet A to blue radiations. In this review, studies on the effects of violet and blue lights emitted by low-power LEDs on bacteria were accessed by PubMed, and discussed the repair of ultraviolet-induced DNA damage by photoreactivation mechanisms. Data from such studies suggested bacterial inactivation after exposure to violet (405 nm) and blue (425-460 nm) radiations emitted from LEDs. However, other studies showed bacterial photoreactivation induced by radiations at 348-440 nm. This process occurs by photolyase enzymes, which absorb photons at wavelengths and repair DNA damage. Although authors have reported bacterial inactivation after exposure to violet and blue radiations emitted from LEDs, pre-exposure to such radiations at low fluences could activate the photolyases, increasing resistance to DNA damage induced by ultraviolet radiation.

A deazariboflavin chromophore kinetically stabilizes reduced FAD state in a bifunctional cryptochrome.

An animal-like cryptochrome derived from Chlamydomonas reinhardtii (CraCRY) is a bifunctional flavoenzyme harboring flavin adenine dinucleotide (FAD) as a photoreceptive/catalytic center and functions both in the regulation of gene transcription and the repair of UV-induced DNA lesions in a light-dependent manner, using different FAD redox states. To address how CraCRY stabilizes the physiologically relevant redox state of FAD, we investigated the thermodynamic and kinetic stability of the two-electron reduced anionic FAD state (FADH-) in CraCRY and related (6-4) photolyases. The thermodynamic stability of FADH- remained almost the same compared to that of all tested proteins. However, the kinetic stability of FADH- varied remarkably depending on the local structure of the secondary pocket, where an auxiliary chromophore, 8-hydroxy-7,8-didemethyl-5-deazariboflavin (8-HDF), can be accommodated. The observed effect of 8-HDF uptake on the enhancement of the kinetic stability of FADH- suggests an essential role of 8-HDF in the bifunctionality of CraCRY.

Molecular Bases of Signaling Processes Regulated by Cryptochrome Sensory Photoreceptors in Plants.

The blue-light sensors, cryptochromes, compose the extensive class of flavoprotein photoreceptors, regulating signaling processes in plants underlying their development, growth, and metabolism. In several algae, cryptochromes may act not only as sensory photoreceptors but also as photolyases, catalyzing repair of the UV-induced DNA lesions. Cryptochromes bind FAD as the chromophore at the photolyase homologous region (PHR) domain and contain the cryptochrome C-terminal extension (CCE), which is absent in photolyases. Photosensory process in cryptochrome is initiated by photochemical chromophore conversions, including formation of the FAD redox forms. In the state with the chromophore reduced to neutral radical (FADH×), the photoreceptor protein undergoes phosphorylation, conformational changes, and disengagement from the PHR domain and CCE with subsequent formation of oligomers of cryptochrome molecules. Photooligomerization is a structural basis of the functional activities of cryptochromes, since it ensures formation of their complexes with a variety of signaling proteins, including transcriptional factors and regulators of transcription. Interactions in such complexes change the protein signaling activities, leading to regulation of gene expression and plant photomorphogenesis. In recent years, multiple papers, reporting novel, more detailed information about the molecular mechanisms of above-mentioned processes were published. The present review mainly focuses on analysis of the data contained in these publications, particularly regarding structural aspects of the cryptochrome transitions into photoactivated states and regulatory signaling processes mediated by the cryptochrome photoreceptors in plants.

PHOTOLYASE/BLUE LIGHT RECEPTOR2 regulates chrysanthemum flowering by compensating for gibberellin perception.

The gibberellins (GAs) receptor GA INSENSITIVE DWARF1 (GID1) plays a central role in GA signal perception and transduction. The typical photoperiodic plant chrysanthemum (Chrysanthemum morifolium) only flowers when grown in short-day photoperiods. In addition, chrysanthemum flowering is also controlled by the aging pathway, but whether and how GAs participate in photoperiod- and age-dependent regulation of flowering remain unknown. Here, we demonstrate that photoperiod affects CmGID1B expression in response to GAs and developmental age. Moreover, we identified PHOTOLYASE/BLUE LIGHT RECEPTOR2, an atypical photocleavage synthase, as a CRYPTOCHROME-INTERACTING bHLH1 interactor with which it forms a complex in response to short days to activate CmGID1B transcription. Knocking down CmGID1B raised endogenous bioactive GA contents and GA signal perception, in turn modulating the expression of the aging-related genes MicroRNA156 and SQUAMOSA PROMOTER BINDING PROTEIN-LIKE3. We propose that exposure to short days accelerates the juvenile-to-adult transition by increasing endogenous GA contents and response to GAs, leading to entry into floral transformation.

Divergent roles of Rad4 and Rad23 homologs in Metarhizium robertsii's resistance to solar ultraviolet damage.

The anti-ultraviolet (UV) role of a Rad4-Rad23-Rad33 complex in budding yeast relies on nucleotide excision repair (NER), which is mechanistically distinct from photorepair of DNA lesions generated under solar UV irradiation but remains poorly known in filamentous fungi. Here, two nucleus-specific Rad4 paralogs (Rad4A and Rad4B) and nucleocytoplasmic shuttling Rad23 ortholog are functionally characterized by multiple analyses of their null mutants in Metarhizium robertsii, an entomopathogenic fungus lacking Rad33. Rad4A was proven to interact with Rad23 and contribute significantly more to conidial UVB resistance (90%) than Rad23 (65%). Despite no other biological function, Rad4A exhibited a very high activity in photoreactivation of UVB-impaired/inactivated conidia by 5-h light exposure due to its interaction with Rad10, an anti-UV protein clarified previously to have acquired a similar photoreactivation activity through its interaction with a photolyase in M. robertsii. The NER activity of Rad4A or Rad23 was revealed by lower reactivation rates of moderately impaired conidia after 24-h dark incubation but hardly observable at the end of 12-h dark incubation, suggesting an infeasibility of its NER activity in the field where nighttime is too short. Aside from a remarkable contribution to conidial UVB resistance, Rad23 had pleiotropic effect in radial growth, aerial conidiation, antioxidant response, and cell wall integrity but no photoreactivation activity. However, Rad4B proved redundant in function. The high photoreactivation activity of Rad4A unveils its essentiality for M. robertsii's fitness to solar UV irradiation and is distinct from the yeast homolog's anti-UV role depending on NER. IMPORTANCE Resilience of solar ultraviolet (UV)-impaired cells is crucial for the application of fungal insecticides based on formulated conidia. Anti-UV roles of Rad4, Rad23, and Rad33 rely upon nucleotide excision repair (NER) of DNA lesions in budding yeast. Among two Rad4 paralogs and Rad23 ortholog characterized in Metarhizium robertsii lacking Rad33, Rad4A contributes to conidial UVB resistance more than Rad23, which interacts with Rad4A rather than functionally redundant Rad4B. Rad4A acquires a high activity in photoreactivation of conidia severely impaired or inactivated by UVB irradiation through its interaction with Rad10, another anti-UV protein previously proven to interact with a photorepair-required photolyase. The NER activity of either Rad4A or Rad23 is seemingly extant but unfeasible under field conditions. Rad23 has pleiotropic effect in the asexual cycle in vitro but no photoreactivation activity. Therefore, the strong anti-UV role of Rad4A depends on photoreactivation, unveiling a scenario distinct from the yeast homolog's NER-reliant anti-UV role.

Role of Substrate Binding Interactions on DNA Repair by Photolyase.

The repair of the cyclobutane pyrimidine dimer (CPD) lesion in DNA by photolyase is determined by its initial recognition, and the catalytic efficiency depends on a series of intermolecular electron-transfer (ET) processes. Here, we investigated the repair of a CPD structural isomer, replacing the deoxyribose with a pyranose sugar on the 5' site, and found a loss in binding efficiency and repair quantum yield. Using femtosecond spectroscopy, we characterized all elementary repair steps and observed a systemic slowdown of the four intermolecular ET reactions and the second bond splitting. Our observations and molecular dynamics simulations suggest that the sugar replacement disrupts the lesion binding configuration, weakening the electronic coupling between the cofactor and lesion and altering the stability of lesion intermediates. These findings highlight how the CPD photolyases have utilized the structural features of the CPD lesion and optimized its interactions with the cofactor and key active-site residues to maximize repair yields.

Ultraviolet B-Rays Induced Gene Alterations and DNA Repair Enzymes in Skin Tissue.

BACKGROUND: Ultraviolet (UV) radiation leads to deoxyribonucleic acid (DNA) damage and changes in gene expression. Topical DNA repair enzymes in liposomes are capable of undoing this damage. OBJECTIVE: To evaluate gene expression changes induced by ultraviolent B-rays (UVB) light and assess the effect of topical DNA repair enzymes extracted from Micrococcus luteus (M. luteus) and photolyase in modifying these changes. METHODS: Non-invasive, adhesive patch collection kits were used to sample skin on the right and left post-auricular areas before and 24 hours after UVB exposure (n=48). Subjects applied topical DNA repair enzymes to the right post-auricular area daily for 2 weeks. Subjects returned 2 weeks later for repeat non-invasive skin sample collection. RESULTS: Eight of 18 tested genes demonstrated significant changes 24 hours following UVB exposure. DNA repair enzymes from M. luteus or photolyase had no significant effect on genetic expression compared with the control at 2 weeks post UV exposure. CONCLUSION: UVB exposure causes acute changes in gene expression, which may play roles in photo-aging damage and skin cancer growth and regulation. While non-invasive gene expression testing can detect UV damage, additional genomic studies investigating recovery from UV damage at different time periods are needed to establish the potential of DNA repair enzymes to minimize or reverse this damage. J Drugs Dermatol. 2023;22(5): doi:10.36849/JDD.7070.

CrusTome: a transcriptome database resource for large-scale analyses across Crustacea.

Transcriptomes from nontraditional model organisms often harbor a wealth of unexplored data. Examining these data sets can lead to clarity and novel insights in traditional systems, as well as to discoveries across a multitude of fields. Despite significant advances in DNA sequencing technologies and in their adoption, access to genomic and transcriptomic resources for nontraditional model organisms remains limited. Crustaceans, for example, being among the most numerous, diverse, and widely distributed taxa on the planet, often serve as excellent systems to address ecological, evolutionary, and organismal questions. While they are ubiquitously present across environments, and of economic and food security importance, they remain severely underrepresented in publicly available sequence databases. Here, we present CrusTome, a multispecies, multitissue, transcriptome database of 201 assembled mRNA transcriptomes (189 crustaceans, 30 of which were previously unpublished, and 12 ecdysozoans for phylogenetic context) as an evolving and publicly available resource. This database is suitable for evolutionary, ecological, and functional studies that employ genomic/transcriptomic techniques and data sets. CrusTome is presented in BLAST and DIAMOND formats, providing robust data sets for sequence similarity searches, orthology assignments, phylogenetic inference, etc. and thus allowing for straightforward incorporation into existing custom pipelines for high-throughput analyses. In addition, to illustrate the use and potential of CrusTome, we conducted phylogenetic analyses elucidating the identity and evolution of the cryptochrome/photolyase family of proteins across crustaceans.