RESUMO
UV light is a potent mutagen that induces bulky DNA damage in the form of cyclobutane pyrimidine dimers (CPDs). Photodamage and other bulky lesions occurring in nuclear genomes can be repaired through nucleotide excision repair (NER), where incisions on both sides of a damaged site precede the removal of a single-stranded oligonucleotide containing the damage. Mitochondrial genomes (mtDNAs) are also susceptible to damage from UV light, but current evidence suggests that the only way to eliminate bulky mtDNA damage is through mtDNA degradation. Damage-containing oligonucleotides excised during NER can be captured with antidamage antibodies and sequenced (XR-seq) to produce high-resolution maps of active repair locations following UV exposure. We analyzed previously published datasets from Arabidopsis thaliana, Saccharomyces cerevisiae, and Drosophila melanogaster to identify reads originating from the mtDNA (and plastid genome in A. thaliana). In A. thaliana and S. cerevisiae, the mtDNA-mapping reads have unique length distributions compared to the nuclear-mapping reads. The dominant fragment size was 26 nt in S. cerevisiae and 28 nt in A. thaliana with distinct secondary peaks occurring in regular intervals. These reads also show a nonrandom distribution of di-pyrimidines (the substrate for CPD formation) with TT enrichment at positions 7-8 of the reads. Therefore, UV damage to mtDNA appears to result in production of DNA fragments of characteristic lengths and positions relative to the damaged location. The mechanisms producing these fragments are unclear, but we hypothesize that they result from a previously uncharacterized DNA degradation pathway or repair mechanism in mitochondria.
Assuntos
Arabidopsis , Dano ao DNA , Reparo do DNA , DNA Mitocondrial , Drosophila melanogaster , Saccharomyces cerevisiae , Raios Ultravioleta , DNA Mitocondrial/genética , Arabidopsis/genética , Arabidopsis/efeitos da radiação , Raios Ultravioleta/efeitos adversos , Animais , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/efeitos da radiação , Saccharomyces cerevisiae/metabolismo , Drosophila melanogaster/genética , Dímeros de Pirimidina/genética , Dímeros de Pirimidina/metabolismo , Genoma MitocondrialRESUMO
This work investigates the feasibility of yeast-based impedance measurements for retrospective dosimetry applications. The local environment around yeast cells in a previously developed film-badge was modeled using Geant4. A greater dose response was observed when yeast cells were surrounded by an aluminum-polymer structure, which acted as a conversion layer. Bench-top experiments were conducted using a jar-based dosimeter design that directly combined a finely-ground aluminum conversion medium with yeast powder. It was shown when irradiated in the presence of aluminum grains, yeast cells yielded a higher impedance signal, thereby indicating greater radiation-induced damage. Finally, in separate irradiation experiments, lead and aluminum sheets were placed behind yeast samples and the dosimeters were irradiated to 1 Gy. A 2-fold increase in the impedance signal was shown when samples were positioned in close contact with the lead sheet compared to the aluminum sheet. In all experiments, it was shown that the local environment significantly influences radiative energy deposition in yeast cells.
Assuntos
Impedância Elétrica , Saccharomyces cerevisiae , Saccharomyces cerevisiae/efeitos da radiação , Alumínio/química , Radiometria/métodos , Radiometria/instrumentação , Doses de Radiação , Dosímetros de RadiaçãoRESUMO
Impedance spectroscopy was employed to assess the electrical properties of yeast following 405 nm laser irradiation, exploring the effects of visible, non-ionizing laser-induced inactivation as a more selective and safer alternative for photoinactivation applications compared to the use of DNA targeting, ionizing UV light. Capacitance and impedance spectra were obtained for yeast suspensions irradiated for 10, 20, 30, and 40 min using 100 and 200 mW laser powers. Noticeable differences in capacitance spectra were observed at lower frequencies (40 Hz to 1 kHz), with a significant increase at 40 min for both laser powers. ß-dispersion was evident in the impedance spectra in the frequency range of 10 kHz to 10 MHz. The characteristic frequency of dielectric relaxation steadily shifted to higher frequencies with increasing irradiation time, with a drastic change observed at 40 min for both laser powers. These changes signify a distinct alteration in the physical state of yeast. A yeast spot assay demonstrated a decrease in cell viability with increasing laser irradiation dose. The results indicate a correlation between changes in electrical properties, cell viability, and the efficacy of 405 nm laser-induced inactivation. Impedance spectroscopy is shown to be an efficient, non-destructive, label-free method for monitoring changes in cell viability in photobiological effect studies. The development of impedance spectroscopy-based real-time studies in photoinactivation holds promise for advancing our understanding of light-cell interactions in medical applications.
Assuntos
Espectroscopia Dielétrica , Lasers , Saccharomyces cerevisiae , Saccharomyces cerevisiae/efeitos da radiação , Viabilidade Microbiana/efeitos da radiaçãoRESUMO
ABSTRACT: Experiments that examine the impacts of subnatural background radiation exposure provide a unique approach to studying the biological effects of low-dose radiation. These experiments often need to be conducted in deep underground laboratories in order to filter surface-level cosmic radiation. This presents some logistical challenges in experimental design and necessitates a model organism with minimal maintenance. As such, desiccated yeast ( Saccharomyces cerevisiae ) is an ideal model system for these investigations. This study aimed to determine the impact of prolonged sub-background radiation exposure in anhydrobiotic (desiccated) yeast at SNOLAB in Sudbury, Ontario, Canada. Two yeast strains were used: a normal wild type and an isogenic recombinational repair-deficient rad51 knockout strain ( rad51 Δ). Desiccated yeast samples were stored in the normal background surface control laboratory (68.0 nGy h -1 ) and in the sub-background environment within SNOLAB (10.1 nGy h -1 ) for up to 48 wk. Post-rehydration survival, growth rate, and metabolic activity were assessed at multiple time points. Survival in the sub-background environment was significantly reduced by a factor of 1.39 and 2.67 in the wild type and rad51 ∆ strains, respectively. Post-rehydration metabolic activity measured via alamarBlue reduction remained unchanged in the wild type strain but was 26% lower in the sub-background rad51 ∆ strain. These results demonstrate that removing natural background radiation negatively impacts the survival and metabolism of desiccated yeast, highlighting the potential importance of natural radiation exposure in maintaining homeostasis of living organisms.
Assuntos
Dessecação , Saccharomyces cerevisiae , Saccharomyces cerevisiae/efeitos da radiação , Rad51 Recombinase/metabolismo , Exposição à Radiação/efeitos adversos , Exposição à Radiação/análise , Doses de RadiaçãoRESUMO
The objectives of this research were to study the effect of UV irradiation on quality characteristics of mango juice during cold storage. Mango juice exposed to UV radiation was also used to determine zero-order and first-order kinetic models of microbial (total plate count, yeast and mold count, and Escherichia coli) reduction. According to the microbiological results, UV light at 120 J/cm2 caused a 5.19 log reduction. It was found that microbial inactivation of all tested microorganisms followed first-order kinetic model. The treatments did not differ significantly in terms of the quality metrics. L*, b*, pH, total soluble solid, total phenolic compound, total flavonoid content, and antioxidant activity as measured by the DPPH and FRAP assay all tended to decline during storage at 4 °C, whereas a*, ∆E, titratable acidity, total plate count, yeast and mold count, as well as the total plate count, had an increasing trend. During storage at 4 °C, UV irradiation increased the shelf life of mango juice by about 14 days compared to the control sample. In conclusion, this study demonstrated the potential of UV treatment as an alternative to thermal pasteurization for preserving mango juice quality and safety while also prolonging shelf life.
Assuntos
Mangifera , Pasteurização , Pasteurização/métodos , Raios Ultravioleta , Saccharomyces cerevisiae/efeitos da radiação , Antioxidantes/análiseRESUMO
Experiment shows ease by which organisms could harness sunlight to produce energy.
Assuntos
Fotossíntese , Rodopsina , Saccharomyces cerevisiae , Luz , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/efeitos da radiação , Luz Solar , Engenharia Genética , Rodopsina/genéticaRESUMO
Ultraviolet-induced DNA lesions impede DNA replication and transcription and are therefore a potential source of genome instability. Here, we performed serial transfer experiments on nucleotide excision repair-deficient (rad14Δ) yeast cells in the presence of chronic low-dose ultraviolet irradiation, focusing on the mechanisms underlying adaptive responses to chronic low-dose ultraviolet irradiation. Our results show that the entire haploid rad14Δ population rapidly becomes diploid during chronic low-dose ultraviolet exposure, and the evolved diploid rad14Δ cells were more chronic low-dose ultraviolet-resistant than haploid cells. Strikingly, single-stranded DNA, but not pyrimidine dimer, accumulation is associated with diploid-dependent fitness in response to chronic low-dose ultraviolet stress, suggesting that efficient repair of single-stranded DNA tracts is beneficial for chronic low-dose ultraviolet tolerance. Consistent with this hypothesis, homologous recombination is essential for the rapid evolutionary adaptation of diploidy, and rad14Δ cells lacking Rad51 recombinase, a key player in homologous recombination, exhibited abnormal cell morphology characterized by multiple RPA-yellow fluorescent protein foci after chronic low-dose ultraviolet exposure. Furthermore, interhomolog recombination is increased in chronic low-dose ultraviolet-exposed rad14Δ diploids, which causes frequent loss of heterozygosity. Thus, our results highlight the importance of homologous recombination in the survival and genomic stability of cells with unrepaired lesions.
Assuntos
Dano ao DNA , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Raios Ultravioleta , Diploide , Reparo do DNA , DNA de Cadeia Simples , Recombinação Homóloga , Rad51 Recombinase/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/efeitos da radiação , Proteínas de Saccharomyces cerevisiae/genética , Adaptação Fisiológica/genéticaRESUMO
The design and implementation of synthetic circuits that operate robustly in the cellular context is fundamental for the advancement of synthetic biology. However, their practical implementation presents challenges due to low predictability of synthetic circuit design and time-intensive troubleshooting. Here, we present the Cyberloop, a testing framework to accelerate the design process and implementation of biomolecular controllers. Cellular fluorescence measurements are sent in real-time to a computer simulating candidate stochastic controllers, which in turn compute the control inputs and feed them back to the controlled cells via light stimulation. Applying this framework to yeast cells engineered with optogenetic tools, we examine and characterize different biomolecular controllers, test the impact of non-ideal circuit behaviors such as dilution on their operation, and qualitatively demonstrate improvements in controller function with certain network modifications. From this analysis, we derive conditions for desirable biomolecular controller performance, thereby avoiding pitfalls during its biological implementation.
Assuntos
Regulação da Expressão Gênica/genética , Optogenética/métodos , Análise de Célula Única/métodos , Processos Estocásticos , Biologia Sintética/métodos , Simulação por Computador , Retroalimentação Fisiológica/efeitos da radiação , Regulação da Expressão Gênica/efeitos da radiação , Luz , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/efeitos da radiaçãoRESUMO
Complex interactions among DNA and nuclear proteins maintain genome organization and stability. The nuclear proteins, particularly the histones, organize, compact, and preserve the stability of DNA, but also allow its dynamic reorganization whenever the nuclear processes require access to it. Five histone classes exist and they are evolutionarily conserved among eukaryotes. The linker histones are the fifth class and over time, their role in chromatin has been neglected. Linker histones interact with DNA and the other histones and thus sustain genome stability and nuclear organization. Saccharomyces cerevisiae is a brilliant model for studying linker histones as the gene for it is a single-copy and is non-essential. We, therefore, created a linker histone-free yeast strain using a knockout of the relevant gene and traced the way cells age chronologically. Here we present our results demonstrating that the altered chromatin dynamics during the chronological lifespan of the yeast cells with a mutation in ARP4 (the actin-related protein 4) and without the gene HHO1 for the linker histone leads to strong alterations in the gene expression profiles of a subset of genes involved in DNA repair and autophagy. The obtained results further prove that the yeast mutants have reduced survival upon UVA/B irradiation possibly due to the accelerated decompaction of chromatin and impaired proliferation. Our hypothesis posits that the higher-order chromatin structure and the interactions among chromatin proteins are crucial for the maintenance of chromatin organization during chronological aging under optimal and UVA-B stress conditions.
Assuntos
Senescência Celular/efeitos da radiação , Cromatina/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/efeitos da radiação , Estresse Fisiológico/efeitos da radiação , Raios Ultravioleta , Ciclo Celular/efeitos da radiação , Relação Dose-Resposta à Radiação , Perfilação da Expressão Gênica , Regulação Fúngica da Expressão Gênica/efeitos da radiação , Histonas/metabolismo , Mutação/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Estresse Fisiológico/genética , Fatores de TempoRESUMO
Microorganisms live in dense and diverse communities, with interactions between cells guiding community development and phenotype. The ability to perturb specific intercellular interactions in space and time provides a powerful route to determining the critical interactions and design rules for microbial communities. Approaches using optogenetic tools to modulate these interactions offer promise, as light can be exquisitely controlled in space and time. We report new plasmids for rapid integration of an optogenetic system into Saccharomyces cerevisiae to engineer light control of expression of a gene of interest. In a proof-of-principle study, we demonstrate the ability to control a model cooperative interaction, namely, the expression of the enzyme invertase (SUC2) which allows S. cerevisiae to hydrolyze sucrose and utilize it as a carbon source. We demonstrate that the strength of this cooperative interaction can be tuned in space and time by modulating light intensity and through spatial control of illumination. Spatial control of light allows cooperators and cheaters to be spatially segregated, and we show that the interplay between cooperative and inhibitory interactions in space can lead to pattern formation. Our strategy can be applied to achieve spatiotemporal control of expression of a gene of interest in S. cerevisiae to perturb both intercellular and interspecies interactions. IMPORTANCE Recent advances in microbial ecology have highlighted the importance of intercellular interactions in controlling the development, composition, and resilience of microbial communities. In order to better understand the role of these interactions in governing community development, it is critical to be able to alter them in a controlled manner. Optogenetically controlled interactions offer advantages over static perturbations or chemically controlled interactions, as light can be manipulated in space and time and does not require the addition of nutrients or antibiotics. Here, we report a system for rapidly achieving light control of a gene of interest in the important model organism Saccharomyces cerevisiae and demonstrate that by controlling expression of the enzyme invertase, we can control cooperative interactions. This approach will be useful for understanding intercellular and interspecies interactions in natural and synthetic microbial consortia containing S. cerevisiae and serves as a proof of principle for implementing this approach in other consortia.
Assuntos
Regulação Fúngica da Expressão Gênica/efeitos da radiação , Luz , Optogenética/métodos , Plasmídeos/genética , Saccharomyces cerevisiae/genética , Estudo de Prova de Conceito , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/efeitos da radiação , Sacarose/metabolismo , beta-Frutofuranosidase/genética , beta-Frutofuranosidase/metabolismoRESUMO
As we explore beyond Earth, astronauts may be at risk for harmful DNA damage caused by ionizing radiation. Double-strand breaks are a type of DNA damage that can be repaired by two major cellular pathways: non-homologous end joining, during which insertions or deletions may be added at the break site, and homologous recombination, in which the DNA sequence often remains unchanged. Previous work suggests that space conditions may impact the choice of DNA repair pathway, potentially compounding the risks of increased radiation exposure during space travel. However, our understanding of this problem has been limited by technical and safety concerns, which have prevented integral study of the DNA repair process in space. The CRISPR/Cas9 gene editing system offers a model for the safe and targeted generation of double-strand breaks in eukaryotes. Here we describe a CRISPR-based assay for DNA break induction and assessment of double-strand break repair pathway choice entirely in space. As necessary steps in this process, we describe the first successful genetic transformation and CRISPR/Cas9 genome editing in space. These milestones represent a significant expansion of the molecular biology toolkit onboard the International Space Station.
Assuntos
Sistemas CRISPR-Cas/genética , Radiação Cósmica/efeitos adversos , Quebras de DNA de Cadeia Dupla/efeitos da radiação , Reparo do DNA/efeitos da radiação , Exposição Ocupacional/efeitos adversos , Astronautas , DNA Fúngico/genética , DNA Fúngico/efeitos da radiação , Edição de Genes , Humanos , Mutagênese , Mutação , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/efeitos da radiação , Proteínas de Saccharomyces cerevisiae/genética , AstronaveRESUMO
SCOPE: The treatment of food with ultraviolet-B (UV-B) light to increase the vitamin D content is accompanied by the formation of photoisomers, such as lumisterol2 . The physiological impact of photoisomers is largely unknown. METHODS AND RESULTS: Three groups of C57Bl/6 mice are fed diets containing 50 µg kg-1 deuterated vitamin D3 with 0, 50 (moderate-dose) or 2000 µg kg-1 (high-dose) lumisterol2 for four weeks. Considerable quantities of lumisterol2 and vitamin D2 are found in the plasma and tissues of mice fed with 2000 µg kg-1 lumisterol2 but not in those fed 0 or 50 µg kg-1 lumisterol2 . Mice fed with 2000 µg kg-1 lumisterol2 showed strongly reduced deuterated 25-hydroxyvitamin D3 (-50%) and calcitriol (-80%) levels in plasma, accompanied by downregulated mRNA abundance of cytochrom P450 (Cyp)27b1 and upregulated Cyp24a1 in the kidneys. Increased tissue levels of vitamin D2 were also seen in mice in a second study that are kept on a diet with 0.2% UV-B exposed yeast versus those fed 0.2% untreated yeast containing iso-amounts of vitamin D2 . CONCLUSION: High doses of lumisterol2 can enter the body, induce the formation of vitamin D2 , reduce the levels of 25(OH)D3 and calcitriol and strongly impact the expression of genes involved in the degradation and synthesis of bioactive vitamin D.
Assuntos
Ergosterol/farmacologia , Vitamina D/metabolismo , 25-Hidroxivitamina D3 1-alfa-Hidroxilase/metabolismo , Administração Oral , Animais , Calcifediol/sangue , Calcitriol/sangue , Dieta , Rim/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Saccharomyces cerevisiae/efeitos da radiação , Raios Ultravioleta , Vitamina D3 24-Hidroxilase/metabolismoRESUMO
Here we describe the development and characterization of the photo-N-degron, a peptide tag that can be used in optogenetic studies of protein function in vivo. The photo-N-degron can be expressed as a genetic fusion to the amino termini of other proteins, where it undergoes a blue light-dependent conformational change that exposes a signal for the class of ubiquitin ligases, the N-recognins, which mediate the N-end rule mechanism of proteasomal degradation. We demonstrate that the photo-N-degron can be used to direct light-mediated degradation of proteins in Saccharomyces cerevisiae and Drosophila melanogaster with fine temporal control. In addition, we compare the effectiveness of the photo-N-degron with that of two other light-dependent degrons that have been developed in their abilities to mediate the loss of function of Cactus, a component of the dorsal-ventral patterning system in the Drosophila embryo. We find that like the photo-N-degron, the blue light-inducible degradation (B-LID) domain, a light-activated degron that must be placed at the carboxy terminus of targeted proteins, is also effective in eliciting light-dependent loss of Cactus function, as determined by embryonic dorsal-ventral patterning phenotypes. In contrast, another previously described photosensitive degron (psd), which also must be located at the carboxy terminus of associated proteins, has little effect on Cactus-dependent phenotypes in response to illumination of developing embryos. These and other observations indicate that care must be taken in the selection and application of light-dependent and other inducible degrons for use in studies of protein function in vivo, but importantly demonstrate that N- and C-terminal fusions to the photo-N-degron and the B-LID domain, respectively, support light-dependent degradation in vivo.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Drosophila melanogaster/efeitos da radiação , Optogenética/métodos , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/efeitos da radiação , Animais , Arginina/metabolismo , Avena , Núcleo Celular/metabolismo , Núcleo Celular/efeitos da radiação , Escuridão , Drosophila melanogaster/embriologia , Embrião não Mamífero/metabolismo , Embrião não Mamífero/efeitos da radiação , Feminino , Fluorescência , Lasers , Luz , Mutação com Perda de Função , Masculino , Proteínas de Neoplasias/metabolismo , Fenótipo , Complexo de Endopeptidases do Proteassoma/metabolismo , Domínios Proteicos/efeitos da radiação , Proteínas Serina-Treonina Quinases/química , Proteólise/efeitos da radiação , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Sunscreens have now been around for decades to mitigate the Sun's damaging ultraviolet (UV) radiation which, although essential for the existence of life, is a recognized prime carcinogen. Accordingly, have suncreams achieved their intended purposes towards protection against sunburns, skin photo-ageing and the like? Most importantly, however, have they provided the expected protection against skin cancers that current sunscreen products claim to do? In the last two decades, there have been tens, if not hundreds of studies on sunscreens with respect to skin protection against UVB (280â320 nm)-traditionally sunscreens with rather low sun protection factors (SPF) were intended to protect against this type of radiation-and UVA (320â400 nm) radiation; a distinction between SPF and UVA protection factor (UVA-PF) is made. Many of the studies of the last two decades have focused on protection against the more skin-penetrating UVA radiation. This non-exhaustive article reviews some of the important facets of what is currently known about sunscreens with regard (i) to the physical UV filters titanium dioxide (TiO2) and zinc oxide (ZnO) and the mostly photo-unstable chemical UVB/UVA filters (e.g., octinoxate (OMC) and avobenzone (AVO), among others), (ii) to novel chemical sunscreen agents, (iii) to means that minimize the breakdown of chemical filters and improve their stability when exposed to UV sunlight, (iv) to SPF factors, and (v) to a short discussion on non-melanoma skin cancers and melanoma. Importantly, throughout the article we allude to the safety aspects of sunscreens and at the end ask the question: do active ingredients in sunscreen products pose a risk to human health, and what else can be done to enhance protection? Significant loss of skin protection from two well-known commercial suncreams when exposed to simulated UV sunlight. Cream I: titanium dioxide, ethylhexyl triazone, avobenzone, and octinoxate; Cream II: octyl salicylate, oxybenzone, avobenzone, and octinoxate.
Assuntos
Pele/efeitos dos fármacos , Protetores Solares/farmacologia , Estabilidade de Medicamentos , Humanos , Propiofenonas/química , Propiofenonas/farmacologia , Propiofenonas/uso terapêutico , Espécies Reativas de Oxigênio/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/efeitos da radiação , Pele/efeitos da radiação , Neoplasias Cutâneas/prevenção & controle , Fator de Proteção Solar , Protetores Solares/química , Protetores Solares/uso terapêutico , Titânio/química , Titânio/farmacologia , Titânio/uso terapêutico , Raios Ultravioleta , Óxido de Zinco/química , Óxido de Zinco/farmacologia , Óxido de Zinco/uso terapêuticoRESUMO
Dynamic control of engineered microbes using light via optogenetics has been demonstrated as an effective strategy for improving the yield of biofuels, chemicals, and other products. An advantage of using light to manipulate microbial metabolism is the relative simplicity of interfacing biological and computer systems, thereby enabling in silico control of the microbe. Using this strategy for control and optimization of product yield requires an understanding of how the microbe responds in real-time to the light inputs. Toward this end, we present mechanistic models of a set of yeast optogenetic circuits. We show how these models can predict short- and long-time response to varying light inputs and how they are amenable to use with model predictive control (the industry standard among advanced control algorithms). These models reveal dynamics characterized by time-scale separation of different circuit components that affect the steady and transient levels of the protein under control of the circuit. Ultimately, this work will help enable real-time control and optimization tools for improving yield and consistency in the production of biofuels and chemicals using microbial fermentations.
Assuntos
Engenharia Metabólica/métodos , Modelos Teóricos , Optogenética/métodos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Algoritmos , Biocombustíveis , Fermentação/efeitos da radiação , Expressão Gênica/efeitos da radiação , Regulação Fúngica da Expressão Gênica/efeitos da radiação , Cinética , Luz , Redes e Vias Metabólicas/efeitos da radiação , Saccharomyces cerevisiae/efeitos da radiaçãoRESUMO
REV3L encodes a catalytic subunit of DNA polymerase zeta (Pol zeta) which is essential for the tolerance of DNA damage by inducing translesion synthesis (TLS). So far, the only Mendelian disease associated with REV3L was Moebius syndrome (3 patients with dominant REV3L mutations causing monoallelic loss-of-function were reported). We describe a homozygous ultra-rare REV3L variant (T2753R) identified with whole exome sequencing in a child without Moebius syndrome but with developmental delay, hypotrophy, and dysmorphic features who was born to healthy parents (heterozygous carriers of the variant). The variant affects the amino acid adjacent to functionally important KKRY motif. By introducing an equivalent mutation (S1192R) into the REV3 gene in yeasts, we showed that, whereas it retained residual function, it caused clear dysfunction of TLS in the nucleus and instability of mitochondrial genetic information. In particular, the mutation increased UV sensitivity measured by cell survival, decreased both the spontaneous (P < 0.005) and UV-induced (P < 0.0001) mutagenesis rates of nuclear DNA and increased the UV-induced mutagenesis rates of mitochondrial DNA (P < 0.0005). We propose that our proband is the first reported case of a REV3L associated disease different from Moebius syndrome both in terms of clinical manifestations and inheritance (autosomal recessive rather than dominant). KEY MESSAGES: First description of a human recessive disorder associated with a REV3L variant. A study in yeast showed that the variant affected the enzymatic function of the protein. In particular, it caused increased UV sensitivity and abnormal mutagenesis rates.
Assuntos
Proteínas de Ligação a DNA/genética , DNA Polimerase Dirigida por DNA/genética , Deficiências do Desenvolvimento/genética , Mutação de Sentido Incorreto , Neoplasias Primárias Múltiplas/genética , Síndromes Neoplásicas Hereditárias/genética , Nevo Pigmentado/genética , Mutação Puntual , Neoplasias Cutâneas/genética , Aldose-Cetose Isomerases/genética , Domínio Catalítico/genética , Pré-Escolar , DNA/metabolismo , DNA Fúngico/genética , DNA Mitocondrial/genética , DNA Mitocondrial/efeitos da radiação , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/fisiologia , DNA Polimerase Dirigida por DNA/química , DNA Polimerase Dirigida por DNA/fisiologia , Deficiências do Desenvolvimento/patologia , Feminino , Homozigoto , Humanos , Masculino , Síndrome de Möbius/genética , Modelos Moleculares , Mutagênese/efeitos da radiação , Linhagem , Fenótipo , Conformação Proteica , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/efeitos da radiação , Proteínas de Saccharomyces cerevisiae/genética , Relação Estrutura-Atividade , Raios Ultravioleta/efeitos adversos , Sequenciamento do ExomaRESUMO
The CMG complex (Cdc45, Mcm2-7, GINS (Psf1, 2, 3, and Sld5)) is crucial for both DNA replication initiation and fork progression. The CMG helicase interaction with the leading strand DNA polymerase epsilon (Pol ε) is essential for the preferential loading of Pol ε onto the leading strand, the stimulation of the polymerase, and the modulation of helicase activity. Here, we analyze the consequences of impaired interaction between Pol ε and GINS in Saccharomyces cerevisiae cells with the psf1-100 mutation. This significantly affects DNA replication activity measured in vitro, while in vivo, the psf1-100 mutation reduces replication fidelity by increasing slippage of Pol ε, which manifests as an elevated number of frameshifts. It also increases the occurrence of single-stranded DNA (ssDNA) gaps and the demand for homologous recombination. The psf1-100 mutant shows elevated recombination rates and synthetic lethality with rad52Δ. Additionally, we observe increased participation of DNA polymerase zeta (Pol ζ) in DNA synthesis. We conclude that the impaired interaction between GINS and Pol ε requires enhanced involvement of error-prone Pol ζ, and increased participation of recombination as a rescue mechanism for recovery of impaired replication forks.
Assuntos
DNA Helicases/metabolismo , DNA Polimerase II/metabolismo , Replicação do DNA/genética , Proteínas Nucleares/metabolismo , Recombinação Genética/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Sobrevivência Celular/genética , Sobrevivência Celular/efeitos da radiação , DNA Polimerase II/genética , Replicação do DNA/efeitos da radiação , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , Mudança da Fase de Leitura do Gene Ribossômico/genética , Mudança da Fase de Leitura do Gene Ribossômico/efeitos da radiação , Pontos de Checagem da Fase G2 do Ciclo Celular/genética , Proteínas de Manutenção de Minicromossomo/metabolismo , Mutagênese , Mutação , Taxa de Mutação , Proteínas Nucleares/genética , Ligação Proteica , Proteína Rad52 de Recombinação e Reparo de DNA/genética , Proteína Rad52 de Recombinação e Reparo de DNA/metabolismo , Recombinação Genética/efeitos da radiação , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/efeitos da radiação , Proteínas de Saccharomyces cerevisiae/genética , Mutações Sintéticas Letais/genéticaRESUMO
UV is a significant environmental agent that damages DNA. Translesion synthesis (TLS) is a DNA damage tolerance pathway that utilizes specialized DNA polymerases to replicate through the damaged DNA, often leading to mutagenesis. In eukaryotic cells, genomic DNA is organized into chromatin that is composed of nucleosomes. To date, if and/or how TLS is regulated by a specific nucleosome feature has been undocumented. We found that mutations of multiple histone H4 residues mostly or entirely embedded in the nucleosomal LRS (loss of ribosomal DNA-silencing) domain attenuate UV mutagenesis in Saccharomyces cerevisiae. The attenuation is not caused by an alteration of ubiquitination or sumoylation of PCNA (proliferating cell nuclear antigen), the modifications well-known to regulate TLS. Also, the attenuation is not caused by decreased chromatin accessibility, or by alterations of methylation of histone H3 K79, which is at the center of the LRS surface. The attenuation may result from compromised TLS by both DNA polymerases ζ and η, in which Rad6 and Rad5 are but Rad18 is not implicated. We propose that a feature of the LRS is recognized or accessed by the TLS machineries either during/after a nucleosome is disassembled in front of a lesion-stalled replication fork, or during/before a nucleosome is reassembled behind a lesion-stalled replication fork.
Assuntos
Histonas/química , Histonas/genética , Mutagênese/genética , Mutagênese/efeitos da radiação , Mutação , Antígeno Nuclear de Célula em Proliferação/metabolismo , Raios Ultravioleta/efeitos adversos , Modelos Moleculares , Domínios Proteicos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/efeitos da radiação , Sumoilação/genética , Sumoilação/efeitos da radiação , Ubiquitinação/genética , Ubiquitinação/efeitos da radiaçãoRESUMO
Exposure to ultraviolet (UV) radiation is the major risk factor for skin cancers. UV induces helix-distorting DNA damage such as cyclobutane pyrimidine dimers (CPDs). If not repaired, CPDs can strongly block DNA and RNA polymerases and cause mutagenesis or cell death. Nucleotide excision repair (NER) is critical for the removal of UV-induced photolesions including CPDs in the cell. Investigating CPD formation and repair across the genome is important for understanding the mechanisms by which these lesions promote somatic mutations in skin cancers. Here we describe a high-throughput, single nucleotide-resolution damage mapping method named CPD sequencing (CPD-seq) for genome-wide analysis of UV-induced CPDs. Protocols for CPD-seq library preparation in yeast and human cells, as well as bioinformatics identification of the CPD damage site, are detailed below.
Assuntos
Mapeamento Cromossômico/métodos , Dano ao DNA/efeitos da radiação , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Dímeros de Pirimidina/genética , Linhagem Celular , Biologia Computacional , Fibroblastos/efeitos da radiação , Humanos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/efeitos da radiação , Neoplasias Cutâneas/genética , Raios UltravioletaRESUMO
Consumer growing demands for high-quality and safe food and beverages have stimulated the interest in alternative preservation technologies. Short-wavelength ultraviolet light (UV-C, 254 nm) has proven to be useful for the decontamination of a great variety of clear juices while improving their quality compared to traditional thermal treatments. Suspended solids and coloured compounds in turbid juices, diminish light transmission. The use of UV-C under a hurdle approach, may be a promising strategy for their treatment. The purpose of this study was to analyse Escherichia coli ATCC 25922, Saccharomyces cerevisiae KE 162 and Lactobacillus plantarum ATCC 8014 inactivation in clear pear juice (PJ), turbid orange-tangerine (OT) and orange-banana-mango-kiwi-strawberry (OBMKS) juices processed by single UV-C (390 mJ/cm2, 20 °C) and UV-C assisted by mild heat (UV-C/H, 50 °C) at pilot-scale in a coiled tubing unit and stored under refrigeration (5 °C). Inactivation studies were also conducted in peptone water (PW) and model solution (MS). The adequacy of the Coroller, Weibull and Biphasic Plus Shoulder models was studied. UV-C was highly effective in PW, MS and PJ, achieving up to 5.5-6.3-4.7, 4.8-5.1-4.6 and 4.4-5.5 log reductions for L. plantarum, E. coli,and S. cerevisiae, respectively. Whereas, a moderate inactivation by single UV-C was recorded in the turbid blends, reducing up to 2.4-3.8-1.6 and 3.6-3.7-1.3 log-cycles in OT and OBMKS, respectively. When the UV-C/H treatment was applied, high bacterial inactivation was observed achieving 5.2-5.6, 6.3-6.6 and 5.5-6.7 log reductions in OT, OBMKS and PJ, respectively, while 4.6-4.9 log reductions were determined for the yeast in OBMKS and OT, respectively. Thus, additive inactivation effects between UV-C and H were observed. All the models tested gave useful information regarding the existence of microbial subpopulations with varying resistances. However, the cumulative Weibull distribution function was the most versatile one, fitting inactivation curves with different shapes. Additionally, the frequency distributions of resistances showed that UV-C/H not only increased the UV-C microbicidal effect but changed the distribution of inactivation times. Principal component analysis revealed that UV-C effectiveness was associated to low particle size, aâ°, turbidity and high UV-C transmittance. An increase on the inactivation of treated bacterial populations was recorded along storage, while no yeast recovery was observed, thus emphasizing the contribution of refrigerated storage to microbial inactivation. Microbial inactivation in clear and turbid juices achieved by UV-C (390 mJ/cm2) assisted by mild heat (50 °C) and subsequent refrigerated storage may represent an useful alternative for multiple applications in the juice industry.