RESUMEN
BACKGROUND: 6 - 4 photoproducts are the second most common UV-induced DNA lesions after cyclobutane pyrimidine dimers. In plants, they are mainly repaired by photolyases in a process called photoreactivation. While pyrimidine dimers can be deleterious, leading to mutagenesis or even cell death, 6 - 4 photoproducts can activate specific signaling pathways. Therefore, their removal is particularly important, especially for plants exposed to high UV intensities due to their sessile nature. Although photoreactivation in nuclear DNA is well-known, its role in plant organelles remains unclear. In this paper we analyzed the activity and localization of GFP-tagged AtUVR3, the 6 - 4 photoproduct specific photolyase. RESULTS: Using transgenic Arabidopsis with different expression levels of AtUVR3, we confirmed a positive trend between these levels and the rate of 6 - 4 photoproduct removal under blue light. Measurements of 6 - 4 photoproduct levels in chloroplast and nuclear DNA of wild type, photolyase mutants, and transgenic plants overexpressing AtUVR3 showed that the photoreactivation is the main repair pathway responsible for the removal of these lesions in both organelles. The GFP-tagged AtUVR3 was predominantly located in nuclei with a small fraction present in chloroplasts and mitochondria of transgenic Arabidopsis thaliana and Nicotiana tabacum lines. In chloroplasts, this photolyase co-localized with the nucleoid marked by plastid envelope DNA binding protein. CONCLUSIONS: Photolyases are mainly localized in plant nuclei, with only a small fraction present in chloroplasts and mitochondria. Despite this unbalanced distribution, photoreactivation is the primary mechanism responsible for the removal of 6 - 4 photoproducts from nuclear and chloroplast DNA in adult leaves. The amount of the AtUVR3 photolyase is the limiting factor influencing the photoreactivation rate of 6 - 4 photoproducts. The efficient photoreactivation of 6 - 4 photoproducts in 35S: AtUVR3-GFP Arabidopsis and Nicotiana tabacum is a promising starting point to evaluate whether transgenic crops overproducing this photolyase are more tolerant to high UV irradiation and how they respond to other abiotic and biotic stresses under field conditions.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Núcleo Celular , Reparación del ADN , Desoxirribodipirimidina Fotoliasa , Plantas Modificadas Genéticamente , Arabidopsis/genética , Arabidopsis/efectos de la radiación , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Núcleo Celular/metabolismo , Núcleo Celular/efectos de la radiación , Desoxirribodipirimidina Fotoliasa/metabolismo , Desoxirribodipirimidina Fotoliasa/genética , Rayos Ultravioleta , ADN de Plantas/metabolismo , ADN de Plantas/genética , Dímeros de Pirimidina/metabolismo , Dímeros de Pirimidina/genética , ADN de Cloroplastos/genética , ADN de Cloroplastos/metabolismo , Cloroplastos/metabolismo , Daño del ADNRESUMEN
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.
Asunto(s)
Núcleo Celular , Daño del ADN , Reparación del ADN , Desoxirribodipirimidina Fotoliasa , Dímeros de Pirimidina , Proteínas Recombinantes , Rayos Ultravioleta , Humanos , Desoxirribodipirimidina Fotoliasa/metabolismo , Desoxirribodipirimidina Fotoliasa/genética , Núcleo Celular/metabolismo , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/genética , Dímeros de Pirimidina/metabolismo , Dímeros de Pirimidina/genética , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/genéticaRESUMEN
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 (ðððð¥ ) of 2.0 g L-1 was reached after 5 h of cultivation, corresponding to ðX = 0.4 g L-1 h. The µððð¥ 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.
Asunto(s)
Desoxirribodipirimidina Fotoliasa , Desoxirribodipirimidina Fotoliasa/genética , Desoxirribodipirimidina Fotoliasa/química , Desoxirribodipirimidina Fotoliasa/metabolismo , Thermus thermophilus , Rayos Ultravioleta , ADN/química , Reparación del ADNRESUMEN
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.
Asunto(s)
Cloroplastos , Desoxirribodipirimidina Fotoliasa , Oryza , Rayos Ultravioleta , Cloroplastos/metabolismo , Desoxirribodipirimidina Fotoliasa/metabolismo , Desoxirribodipirimidina Fotoliasa/genética , Oryza/genética , Oryza/enzimología , Oryza/efectos de la radiación , Oryza/metabolismo , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética , Dímeros de Pirimidina/metabolismo , Poaceae/genética , Poaceae/enzimología , Poaceae/efectos de la radiación , Poaceae/metabolismo , Secuencia de Aminoácidos , Transporte de ProteínasRESUMEN
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.
Asunto(s)
Desoxirribodipirimidina Fotoliasa , Humanos , Animales , Desoxirribodipirimidina Fotoliasa/química , Desoxirribodipirimidina Fotoliasa/genética , Desoxirribodipirimidina Fotoliasa/metabolismo , Triptófano/química , Electrones , Drosophila melanogaster/metabolismo , Escherichia coli/genética , Transporte de Electrón , Cristalografía por Rayos XRESUMEN
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.
Asunto(s)
Desoxirribodipirimidina Fotoliasa , Metarhizium , Rhipicephalus , Animales , Rhipicephalus/metabolismo , Rhipicephalus/microbiología , Desoxirribodipirimidina Fotoliasa/genética , Desoxirribodipirimidina Fotoliasa/metabolismo , Virulencia , Luz , Rayos Ultravioleta , Metarhizium/metabolismo , Control Biológico de VectoresRESUMEN
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.
Asunto(s)
Criptocromos , Desoxirribodipirimidina Fotoliasa , Diatomeas , Criptocromos/química , Criptocromos/genética , Desoxirribodipirimidina Fotoliasa/química , Desoxirribodipirimidina Fotoliasa/genética , Deuterio , Diatomeas/enzimología , Transporte de Electrón , Dominios ProteicosRESUMEN
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 â¼90% 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.
Asunto(s)
Desoxirribodipirimidina Fotoliasa , Metarhizium , Reparación del ADN , Desoxirribodipirimidina Fotoliasa/genética , Desoxirribodipirimidina Fotoliasa/metabolismo , Saccharomyces cerevisiae/genética , Daño del ADN , Metarhizium/genética , Metarhizium/metabolismo , Rayos UltravioletaRESUMEN
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.
Asunto(s)
Chrysanthemum , Desoxirribodipirimidina Fotoliasa , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Giberelinas/metabolismo , Desoxirribodipirimidina Fotoliasa/genética , Desoxirribodipirimidina Fotoliasa/metabolismo , Chrysanthemum/genética , Chrysanthemum/metabolismo , Flores/fisiología , Fotoperiodo , Percepción , Regulación de la Expresión Génica de las PlantasRESUMEN
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.
Asunto(s)
Desoxirribodipirimidina Fotoliasa , Metarhizium , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Desoxirribodipirimidina Fotoliasa/genética , Desoxirribodipirimidina Fotoliasa/metabolismo , Reparación del ADN , Proteínas de Saccharomyces cerevisiae/genética , Metarhizium/genética , Metarhizium/metabolismo , Rayos Ultravioleta , ADN/metabolismo , Proteínas de Unión al ADN/metabolismoRESUMEN
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.
Asunto(s)
Crustáceos , Transcriptoma , Crustáceos/genética , Animales , Desoxirribodipirimidina Fotoliasa/genética , Criptocromos/genética , Filogenia , GenomaRESUMEN
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.
Asunto(s)
Desoxirribodipirimidina Fotoliasa , Neoplasias Cutáneas , Humanos , Daño del ADN , Desoxirribodipirimidina Fotoliasa/genética , Reparación del ADN , Piel/efectos de la radiación , Rayos Ultravioleta/efectos adversos , Enzimas Reparadoras del ADN/genéticaRESUMEN
In bacterial endospores, a cross-linked thymine dimer, 5-thyminyl-5,6-dihydrothymine, commonly referred to as the spore photoproduct (SP), is found as the dominant DNA photo lesion under UV radiation. During spore germination, SP is faithfully repaired by the spore photoproduct lyase (SPL) for normal DNA replication to resume. Despite this general mechanism, the exact way in which SP modifies the duplex DNA structure so that the damaged site can be recognized by SPL to initiate the repair process is still unclear. A previous X-ray crystallographic study, which used a reverse transcriptase as a DNA host template, captured a protein-bound duplex oligonucleotide containing two SP lesions; the study showed shortened hydrogen bonds between the AT base pairs involved in the lesions and widened minor grooves near the damaged sites. However, it remains to be determined whether the results accurately reflect the conformation of SP-containing DNA (SP-DNA) in its fully hydrated pre-repair form. To uncover the intrinsic changes in DNA conformation caused by SP lesions, we performed molecular dynamics (MD) simulations of SP-DNA duplexes in aqueous solution, using the nucleic acid portion of the previously determined crystal structure as a template. After MD relaxation, our simulated SP-DNAs showed weakened hydrogen bonds at the damaged sites compared to those in the undamaged DNA. Our analyses of the MD trajectories revealed a range of local and global structural distortions of DNA induced by SP. Specifically, the SP region displays a greater tendency to adopt an A-like-DNA conformation, and curvature analysis revealed an increase in the global bending compared to the canonical B-DNA. Although these SP-induced DNA conformational changes are relatively minor, they may provide a sufficient structural basis for SP to be recognized by SPL during the lesion repair process.
Asunto(s)
Reparación del ADN , Desoxirribodipirimidina Fotoliasa , Desoxirribodipirimidina Fotoliasa/química , Desoxirribodipirimidina Fotoliasa/genética , Desoxirribodipirimidina Fotoliasa/metabolismo , Simulación por Computador , ADN Bacteriano/genética , ADN , Esporas Bacterianas/genética , Esporas Bacterianas/metabolismo , Rayos UltravioletaRESUMEN
Ultraviolet B (UVB) in sunlight cause skin damage, ranging from wrinkles to photoaging and skin cancer. UVB can affect genomic DNA by creating cyclobutane pyrimidine dimers (CPDs) and pyrimidine-pyrimidine (6-4) photoproducts (6-4PPs). These lesions are mainly repaired by the nucleotide excision repair (NER) system and by photolyase enzymes that are activated by blue light. Our main goal was to validate the use of Xenopus laevis as an in vivo model system for investigating the impact of UVB on skin physiology. The mRNA expression levels of xpc and six other genes of the NER system and CPD/6-4PP photolyases were found at all stages of embryonic development and in all adult tissues tested. When examining Xenopus embryos at different time points after UVB irradiation, we observed a gradual decrease in CPD levels and an increased number of apoptotic cells, together with an epidermal thickening and an increased dendricity of melanocytes. We observed a quick removal of CPDs when embryos are exposed to blue light versus in the dark, confirming the efficient activation of photolyases. A decrease in the number of apoptotic cells and an accelerated return to normal proliferation rate was noted in blue light-exposed embryos compared with their control counterparts. Overall, a gradual decrease in CPD levels, detection of apoptotic cells, thickening of epidermis, and increased dendricity of melanocytes, emulate human skin responses to UVB and support Xenopus as an appropriate and alternative model for such studies.
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Daño del ADN , Desoxirribodipirimidina Fotoliasa , Animales , Humanos , Xenopus laevis/metabolismo , Desoxirribodipirimidina Fotoliasa/genética , Desoxirribodipirimidina Fotoliasa/metabolismo , Dímeros de Pirimidina/genética , Dímeros de Pirimidina/metabolismo , Rayos Ultravioleta/efectos adversosRESUMEN
The cryptochrome/photolyase (CRY/PL) family is essential for life under sunlight because photolyases repair UV-damaged DNA and cryptochromes are normally part of the circadian clock that controls the activity-sleep cycle within the 24-h day. In this study, we aim to understand how the lineage and habitat of an insect affects its CRY/PL composition. To this end, we searched the large number of annotated protein sequences of 340 insect species already available in databases for CRY/PLs. Using phylogenetic tree and motif analyses, we identified four frequent CRY/PLs in insects: the photolyases 6-4 PL and CPDII PL, as well as the mammalian-type cryptochrome (MCRY) and Drosophila-type cryptochrome (DCRY). Assignment of CRY/PLs to the corresponding insects confirmed that light-exposed insects tend to have more CRY/PLs than insects with little light exposure. Nevertheless, even insects with greatly reduced CRY/PLs still possess MCRY, which can be regarded as the major insect cryptochrome. Only flies of the genus Schizophora, which includes Drosophila melanogaster, lost MCRY. Moreover, we found that MCRY and CPDII PL as well as DCRY and 6-4 PL occur very frequently together, suggesting an interaction between the two pairs.
Asunto(s)
Desoxirribodipirimidina Fotoliasa , Proteínas de Drosophila , Animales , Criptocromos/genética , Criptocromos/metabolismo , Desoxirribodipirimidina Fotoliasa/genética , Desoxirribodipirimidina Fotoliasa/metabolismo , Drosophila melanogaster/metabolismo , Luz Solar , Filogenia , Drosophila/metabolismo , Ritmo Circadiano , Proteínas de Drosophila/genética , Mamíferos/metabolismoRESUMEN
Photolyases (PLs) reverse UV-induced DNA damage using blue light as an energy source. Of these PLs, (6-4) PLs repair (6-4)-lesioned photoproducts. We recently identified a gene from Vibrio cholerae (Vc) encoding a (6-4) PL, but structural characterization is needed to elucidate specific interactions with the chromophore cofactors. Here, we determined the crystal structure of Vc (6-4) PL at 2.5 Å resolution. Our high-resolution structure revealed that the two well-known cofactors, flavin adenine dinucleotide and the photoantenna 6,7-dimethyl 8-ribityl-lumazin (DMRL), stably interact with an α-helical and an α/ß domain, respectively. Additionally, the structure has a third cofactor with distinct electron clouds corresponding to a [4Fe-4S] cluster. Moreover, we identified that Asp106 makes a hydrogen bond with water and DMRL, which indicates further stabilization of the photoantenna DMRL within Vc (6-4) PL. Further analysis of the Vc (6-4) PL structure revealed a possible region responsible for DNA binding. The region located between residues 478 to 484 may bind the lesioned DNA, with Arg483 potentially forming a salt bridge with DNA to stabilize further the interaction of Vc (6-4) PL with its substrate. Our comparative analysis revealed that the DNA lesion could not bind to the Vc (6-4) PL in a similar fashion to the Drosophila melanogaster (Dm, (6-4)) PL without a significant conformational change of the protein. The 23rd helix of the bacterial (6-4) PLs seems to have remarkable plasticity, and conformational changes facilitate DNA binding. In conclusion, our structure provides further insight into DNA repair by a (6-4) PL containing three cofactors.
Asunto(s)
Desoxirribodipirimidina Fotoliasa , Vibrio cholerae , Animales , Desoxirribodipirimidina Fotoliasa/genética , Desoxirribodipirimidina Fotoliasa/química , Desoxirribodipirimidina Fotoliasa/metabolismo , Vibrio cholerae/genética , Vibrio cholerae/metabolismo , Drosophila melanogaster/metabolismo , Reparación del ADN , ADN/química , Flavina-Adenina Dinucleótido/metabolismoRESUMEN
One of the most important factor that affects the efficient using of baculoviruses as a biopesticide is their sensitivity to UV irradiation. In this study, a photolyase gene (phr) of 1.4 kbp DNA fragment was cloned and characterized from Spodoptera littoralis granulovirus, an Egyptian isolate (SpliGV-EG1). A sequence of 466 amino acid were deduced when the gene was completely sequenced with a predicted molecular mass of ~ 55 kDa. Transcriptional regulation analyses revealed that phr transcripts were detected early at 6-h post-infection (hpi) and remained detectable until 72 hpi, suggesting their transcriptional regulation from a putative early promoter motif. An approximately ~ 55 kDa protein fragment was expressed from phr-induced bacterial culture and detected by SDS-PAGE and western blotting. In addition, direct exposure to UV irradiation resulted in a twofold decrease in SpliGV-EG1 occlusion bodies activation compared with Spodoptera littoralis nucleopolyhedrovirus (SpliNPV) occlusion bodies which decreased with about 129-fold after exposure to UV irradiation based on median lethal concentration value (LC50). The obtained results suggested that the presence of photolyase gene possibly alters the inactivation of SpliGV-EG1-occluded bodies by UV irradiation. These results support the role and application of the photolyase protein to improve the damaged DNA repair mechanism as well as resistance of SpliGV to UV light inactivation.
Asunto(s)
Desoxirribodipirimidina Fotoliasa , Granulovirus , Animales , Granulovirus/genética , Desoxirribodipirimidina Fotoliasa/genética , Spodoptera/genética , Baculoviridae/genética , Regiones Promotoras GenéticasRESUMEN
UVB radiation is known to trigger the block of DNA replication and transcription by forming cyclobutane pyrimidine dimer (CPD), which results in severe skin damage. CPD photolyase, a kind of DNA repair enzyme, can efficiently repair CPDs that are absent in humans and mice. Although exogenous CPD photolyases have beneficial effects on skin diseases, the mechanisms of CPD photolyases on the skin remain unknown. Here, this study prepared CPD photolyase nanoliposomes (CPDNL) from Antarctic Chlamydomonas sp. ICE-L, which thrives in harsh, high-UVB conditions, and evaluated their protective mechanisms against UVB-induced damage in mice. CPDNL were optimized using response surface methodology, characterized by a mean particle size of 105.5 nm, with an encapsulation efficiency of 63.3%. Topical application of CPDNL prevented UVB-induced erythema, epidermal thickness, and wrinkles in mice. CPDNL mitigated UVB-induced DNA damage by significantly decreasing the CPD concentration. CPDNL exhibited antioxidant properties as they reduced the production of reactive oxygen species (ROS) and malondialdehyde. Through activation of the NF-κB pathway, CPDNL reduced the expression of pro-inflammatory cytokines including IL-6, TNF-α, and COX-2. Furthermore, CPDNL suppressed the MAPK signaling activation by downregulating the mRNA and protein expression of ERK, JNK, and p38 as well as AP-1. The MMP-1 and MMP-2 expressions were also remarkably decreased, which inhibited the collagen degradation. Therefore, we concluded that CPDNL exerted DNA repair, antioxidant, anti-inflammation, and anti-wrinkle properties as well as collagen protection via regulation of the NF-κB/MAPK/MMP signaling pathways in UVB-induced mice, demonstrating that Antarctic CPD photolyases have the potential for skincare products against UVB and photoaging.
Asunto(s)
Desoxirribodipirimidina Fotoliasa , Microalgas , Animales , Humanos , Ratones , Antioxidantes/farmacología , Desoxirribodipirimidina Fotoliasa/genética , Desoxirribodipirimidina Fotoliasa/metabolismo , Daño del ADN , Microalgas/metabolismo , FN-kappa B/genética , Dímeros de Pirimidina/metabolismo , Rayos UltravioletaRESUMEN
Exposure to ultraviolet radiation from sunlight induces oxidative DNA lesions and bipyrimidine photoproducts that can lead to photo-aging and skin carcinogenesis. CPD-photolyases are flavoproteins that repair cyclobutane pyrimidine dimers using blue light as an energy source. In the present work, we evaluated the photo-repair effect of the recombinant CPD-photolyase PhrAHym from the Antarctic bacterium Hymenobacter sp. UV11 on DNA lesions in human keratinocytes induced by UVC light. By performing immunochemistry assays we observed that PhrAHym repairs in a highly efficient way the CPD-photoproducts and reduces the γH2AX formation. Since this enzyme is non-cytotoxic and repairs UVC-induced DNA lesions in human keratinocytes, we propose that PhrAHym could be used as a biotherapeutic agent against UV-induced skin cancer, photoaging, and related diseases.
Asunto(s)
Daño del ADN , Desoxirribodipirimidina Fotoliasa , Queratinocitos , Humanos , Bacterias/enzimología , Bacterias/genética , Desoxirribodipirimidina Fotoliasa/genética , Desoxirribodipirimidina Fotoliasa/metabolismo , Reparación del ADN , Queratinocitos/metabolismo , Queratinocitos/efectos de la radiación , Rayos Ultravioleta/efectos adversosRESUMEN
Light quality is a significant factor for living organisms that have photosensory systems, such as rhodopsin, a seven alpha-helical transmembrane protein with the retinal chromophore. Here, we report, for the first time, the function of new rhodopsin, which is an inverted 7-transmembrane protein, isolated from Trichococcus flocculiformis. T. flocculiformis heliorhodopsin (TfHeR) works as a regulatory helper rhodopsin that binds with class 2 cyclobutane pyrimidine dimer (CPDII) photolyase to broaden the spectrum and upregulate DNA repair activity. We have confirmed their interaction through isothermal titration calorimetry (dissociation constant of 21.7 µM) and identified the charged residues for the interaction. Based on in vivo and in vitro experiments, we showed that the binding of heliorhodopsin with photolyase improved photolyase activity by about 3-fold to repair UV-caused DNA damage. Also, the DNA repair activity of TfHeR/T. flocculiformis photolyase (TfPHR) was observed in the presence of green light. Our results suggested that heliorhodopsin directly controls the activity of photolyase and coevolves to broaden the activity spectrum by protein-protein interaction. IMPORTANCE This study reports a function for Heliorhodopsin working as a regulatory helper rhodopsin that with CPDII photolyase to broaden the spectrum and upregulating the DNA repair activity. Our results suggested that heliorhodopsin directly controls photolyase activity and coevolves to broaden the DNA repair capacity by protein-protein interaction.