The role of reactive oxygen species in the retrograde chloroplast-nucleus signalling pathway

Photosynthetic organisms are particularly susceptible to photooxidative stress, because they are dependent on light energy for converting carbon dioxide into organic compounds and as a by-product, generate high levels of oxygen in the chloroplast. While photosynthetic eukaryotes exhibit altered nuclear gene expression in response to changes in the production of reactive oxygen species (ROS) in the chloroplast, little is known about how this signal is transmitted from the chloroplast to the nucleus. In the green alga Chlamydomonas reinhardtii, the cytosolic GLUTATHIONE PEROXIDASE 5 gene (GPX5) is known to be up-regulated at the level of transcription in response to singlet oxygen. Previous studies have shown that when the promoter region of GPX5 is fused to the ARYLSULFATASE 2 gene (ARS2), an effective reporter system can be generated and used to study GPX5 expression. This system was used in this study to generate a stably transformed C. reinhardtii strain which expresses the ARS2 protein in a singlet oxygen-dependent manner. Using the strain of C. reinhardtii harbouring a singlet oxygen-sensitive promoter gene, secondary mutagenic screen was performed. This allowed identification of mutant cell lines that were unable to up-regulate GPX5-ARS2 fusion expression, based on ARS2 activity, in response to singlet oxygen production. In one of these lines, the mutation was subsequently localized to the first exon of the PSBP-like gene (PSBP2), and this line was designated as psbP2. The PSBP2 gene is part of a small gene family in C. reinhardtii that is conserved in higher plant species. While each member of the PSBP protein family contains a similar domain to the PSBP1 protein, which is a member of the oxygen evolving complex of photosystem II (PSII), the PSBP2 protein does not appear to be involved in PSII function. While psbP2 does not produce greater ARS2 activity in response to singlet oxygen, it still accumulates both the GPX5-ARS2 and native GPX5 transcripts when challenged by photosensitizer exposure, although at lower levels than the original lines. It was demonstrated that the PSBP2 protein is involved in transmitting information related to the accumulation of singlet oxygen in the chloroplast to control the singlet oxygen-dependent GPX5 driven ARS2 expression in the nucleus and/or ARS2 activity through a post-transcriptional process in the cytoplasm

Photoreactivity of DNA Etheno Adducts: Spectroscopic and Mechanistic Study

[ES] Los estudios sobre los daños en el ADN se han incrementado en las últimas décadas con el fin de profundizar en su implicación en la aparición del cáncer. Entre el gran número de lesiones del ADN, los aductos de tipo eteno han sido objeto de interés debido a su presencia en los tejidos humanos crónicamente inflamados. Asimismo, su cuantificación es útil para su uso como potencial biomarcador del cáncer de colon, próstata, pulmón, etc. Además, estas lesiones presentan propiedades altamente mutagénicas e inducen transiciones de bases o transversiones en las células de mamíferos. Los aductos de tipo eteno se generan principalmente de forma endógena como resultado de la peroxidación lipídica. Este proceso bioquímico produce aldehídos reactivos como el malondialdehído, que pueden combinarse con las bases del ADN creando un anillo exocíclico.Este anillo exocíclico proporciona a la nucleobase un sistema extendido p-conjugado que puede conferirles propiedades ópticas diferentes a las de las bases canónicas, lo que puede suponer una amenaza para la fotoestabilidad del ADN. Las bases canónicas tienen la capacidad de disipar la mayor parte de la energía recibida a través de canales no radiativos eficientes que conducen de nuevo al estado fundamental. Por lo tanto, los estudios sobre las propiedades ópticas de estos aductos etenos son clave para establecer si estas lesiones pueden poner en peligro la relajación eficiente de los estados excitados de las bases y desencadenar una fotorreactividad indeseada del ADN. La primera parte de la tesis trata de evaluar la fotoactividad potencial de estas lesiones del ADN mediante un estudio espectroscópico. En el Capítulo 3 se combinan experimentos de upconversión de fluorescencia en la escala de femtosegundos y cálculos teóricos (en los niveles PCM-TD-DFT y CASPT2/CASSCF) para proporcionar una imagen completa de la relajación de los estados excitados del aducto mutagénico 3,N4-eteno-2'-desoxicitidina (edC). El Capítulo 4 aborda las propiedades fotofísicas de los aductos junto con su fotorreactividad en presencia de fotosensibilizadores comunes como la Rosa de Bengala (RB) y la 4-carboxibenzofenona (CBP), prestando especial atención a la interacción con el 1O2. En condiciones aeróbicas, se observa una interacción con 1O2 para los tres aductos estudiados. Curiosamente, se observan los mismos fotoproductos, las nucleobases originales, para la irradiación en condiciones anaeróbicas, abriendo la posibilidad de un mecanismo mixto de Tipo I y Tipo II cuando se utiliza Rosa de Bengala como fotosensibilizador, y de Tipo I para la 4-carboxibenzofenona. Finalmente, el último capítulo trata de unir todos los conocimientos adquiridos sobre la fotorreactividad de los e-aductos para elegir el mejor cromóforo tratando de optimizar el proceso de reparación observado en el Capítulo 4. Para ello se utilizan sistemas híbridos de nanopartículas metálicas de Ag como matriz de soporte para la Rosa de Bengala. Las NPs metálicas, como las Ag NPs, poseen una resonancia plasmónica superficial localizada (LSPR), este efecto amplifica una gran variedad de fenómenos ópticos que pueden mejorar las propiedades ópticas de la Rosa de Bengala.[CA] Els estudis sobre els danys en l'ADN s'han incrementat en les últimes dècades amb la finalitat d'aprofundir en la seua implicació en l'aparició del càncer. Entre el gran nombre de lesions de l'ADN, els adductes de tipus eteno han sigut objecte d'interés degut a la seua presència en els teixits humans crònicament inflamats, la qual cosa fa que la seua quantificació siga útil com a potencials biomarcadors del càncer de còlon, pròstata, pulmó, etc. A més, aquestes lesions presenten propietats altament mutagèniques i indueixen transicions de bases o transversions en les cèl·lules dels mamífers. Els adductes de tipus eteno es formen principalment de manera endògena com a resultat de la peroxidació dels lípids. Aquest procés bioquímic produeix aldehids reactius com el malondialdehid, que poden combinar-se amb les bases de l'ADN creant un anell exocíclic. Aquest anell exocíclic proporciona a les nucleobases un sistema estés p-conjugat que pot conferir-les propietats òptiques diferents a les de les bases canòniques, i que poden suposar una amenaça per a la fotoestabilitat de l'ADN. Les bases canòniques tenen la capacitat de dissipar la major part de l'energia d'excitació a través de canals no radiatius eficients que condueixen de nou a l'estat bàsic. No obstant això, els estudis sobre les propietats òptiques d'aquests adductes de tipus eteno són fonamentals per a deixar clar si aquestes lesions poden posar en perill aquesta relaxació eficient i desencadenar una fotoreactivitat indesitjada de l'ADN. La primera part de la tesi tracta d'estimar el potencial fotoactiu d'aquestes lesions de l'ADN mitjançant un estudi espectroscòpic. En el Capítol 3 es combinen experiments de "upconversió" de fluorescència en una escala de femtosegons i càlculs teòrics (en els nivells PCM-TD-DFT i CASPT2/CASSCF) per a proporcionar una imatge completa de la relaxació dels estats excitats del adducte mutagènic 3,N4-eteno-2'-desoxicitidina (edC). El Capítol 4 aborda les propietats fotofísiques dels adductes restants juntament amb el seua fotoreactivitat en presència d'alguns fotosensibilitzadors comuns com la Rosa de Bengala (RB) i la 4-carboxibenzofenona (CBP), prestant especial atenció a la interacció amb el 1O2. S'observa la interacció amb 1O2 per als tres adductes estudiats. Curiosament, s'observa la mateixa formació de nucleobases per a la irradiació en condicions anaeròbiques, obrint la possibilitat d'un mecanisme mixt de Tipus I i Tipus II quan s'utilitza Rosa de Bengala com fotosensibilitzador i de Tipus I per a la 4-carboxibenzofenona. Finalment, l'últim capítol tracta d'unir tots els coneixements adquirits sobre la fotoreactivitat dels adductes de tipus eteno per a triar el millor cromòfor tractant d'optimitzar el procés de reparació observat en el Capítol 4. Per a això s'utilitzen sistemes híbrids de nanopartícules metàl·liques de Ag com a matriu de suport per a la Rosa de Bengala. Les NPs metàl·liques, com les de Ag, posseeixen ressonància plasmónica superficial localitzada (LSPR), aquest efecte amplifica una gran varietat de fenòmens òptics que poden millorar les propietats òptiques de la Rosa de Bengala.[EN] Studies dealing with DNA damages have increased during the last decades in order to get more insight into their involvement in the appearance of cancer. Among the large number of DNA lesions, etheno adducts have been the matter of interest because of their presence in chronically inflamed human tissues, making their quantification useful as potential biomarkers for cancer of colon, prostate, lung, etc. Moreover, these lesions exhibit highly mutagenic properties and induce base transitions or transversion in mammal cells. Etheno adducts are mainly formed endogenously as a result of lipid peroxidation. This biochemical process produces reactive aldehydes such as malondialdehyde, which can combine with DNA bases creating the exocyclic ring. This exocyclic ring provides to the nucleobases an extended p- conjugated system that might confer them optical properties different from those of the canonical bases, and can pose a threat to the DNA photostability. Canonical bases have the ability to dissipate most of the excitation energy through efficient nonradiative channels leading back to the ground state. However, the studies about the optical properties of this etheno adducts are basics to make it clear whether these lesions can jeopardize this efficient relaxation and trigger undesired DNA photoreactivity. The first part of the thesis establishes the potential photoactivity of these DNA lesions through a spectroscopic study. Chapter 3 joints femtosecond fluorescence upconversion experiments and theoretical calculations (at the PCM-TD-DFT and CASPT2/CASSCF levels) to provide a comprehensive picture of the mutagenic etheno adduct 3,N4-etheno-2'-deoxycytidine (edC) excited states relaxation. Chapter 4 addresses the photophysical properties of the adducts together with its photoreactivity in the presence of some common photosensitizers as Rose Bengal and 4-carboxybenzophenone, paying a special attention to interaction with 1O2. Interaction with 1O2 is observed for the three studied e-adducts. Interestingly, the same nucleobase formation is detected for irradiation under anaerobic conditions, opening the possibility of a mixed Type I and Type II mechanism when Rose Bengal is used as photosensitizer, and Type I for 4-carboxybenzophenone. Finally, the last chapter takes advantage of all the gained knowledge about the photoreactivity of e-adducts to choose the best chromophore and optimize the repair process observed in Chapter 4. To achieve this, hybrid systems of Ag metal nanoparticles are used as a support matrix for the Rose Bengal. Metal NPs, such as Ag NP, possess localized surface plasmon resonance (LSPR). This effect amplifies a wide variety of optical phenomena that can enhance the Rose Bengal optical properties.Lizondo Aranda, P. (2022). Photoreactivity of DNA Etheno Adducts: Spectroscopic and Mechanistic Study [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/18968

Genome mutation and physiology of Synechocystis sp. PCC 6803 wild types and pH-sensitive Photosystem II mutants

The model cyanobacterium Synechocystis sp. PCC 6803 is widely used in studies of photosynthesis, environmental sensing, and stress-response. Its capacity for straightforward genetic engineering and the early publication of its genome sequence meant that substrains of this organism have been dispersed widely among laboratories, particularly for the purposes of investigating the function of the water-splitting enzyme of photosynthesis, Photosystem II (PS II). Recently, advances in genome sequencing technology have revealed genomic divergence among these substrains, with a largely unknown level of resultant phenotypic variation. In this study, the capacity for Synechocystis sp. PCC 6803 wild types to undergo genomic change was analysed by assembly of the genome sequence of the ‘GT-O1’ and ‘GT-O2’ substrains in use at the University of Otago. In the GT-O1 substrain, a possible instance of active genome transposition processes involving a Tc1/mariner-type transposase-encoding gene was observed, and in the GT-O2 substrain a mutation detected in chlH was associated with a reduction in chlorophyll biosynthesis. It is suggested that long-term culture conditions induce genomic changes with major functional consequences in some wild-type substrains, in spite of theoretically ideal laboratory growth conditions. However, phenotypic analysis suggested that the GT-O1 substrain is comparable to other substrains of Synechocystis sp. PCC 6803 held overseas. The capacity for genome mutation in response to gene deletions affecting PS II was also analysed. Some strains carrying mutations in the extrinsic proteins or domains of PS II display an enigmatic pH 7.5-sensitive phenotype, but pH 7.5-growth of a ∆PsbO:∆PsbU strain could be rescued by genome mutations that apparently affect PS II-independent cellular processes. Assembly of the genome of a pH-insensitive ∆PsbO:∆PsbU pseudorevertant identified a mutation in pmgA that appears to affect carbon uptake, and accordingly CO2 enrichment rescued growth of some pH-sensitive PS II mutants, including the ∆PsbO:∆PsbU strain. To further investigate the effect of external pH on the membrane-embedded PS II complex, analysis of a pH-sensitive strain lacking PsbV and carrying a mutation in Loop E of the PS II core antenna CP47 protein revealed that mutations in the vicinity of the redox-active tyrosine YD appear to alter PS II redox equilibria. In a CP47 E364Q:∆PsbV mutant, the stability of YD+QA- charge pairs in PS II and possibly the capacity of YD to maintain charge equilibrium with the PS II oxygen-evolving complex was altered, likely contributing to pH-sensitivity. This suggests that pH affects PS II directly and indirectly, due to a complex interplay of pH effects on electron transport, carbon uptake, pH homeostasis, and PS II redox equilibria

The Reactions in Viral Nucleic Acids During Photolysis and Chlorine Disinfection

Virus-induced diseases pose risks to public health and cause significant impacts on our economy. People can become infected by waterborne virus pathogens when they come into contact with drinking water and recreational water that was not properly treated and disinfected. Nucleic acids (DNA/RNA) carry the genetic instructions for viruses to replicate in their host cells; therefore, damaging viral nucleic acids is an effective way to inactivate viruses and reduce risks of waterborne infection. UV254 and chlorine are two disinfection methods commonly used in water treatment, and both lead to reactions in viral genomes. Despite the widespread use of disinfection, scientists and engineers still lack a comprehensive understanding of the reactions that take place in viral nucleic acids, the impact of higher order structure on viral genome reactivity during UV254 and chlorine disinfection. With this knowledge, it might become possible to predict the inactivation kinetics of newly emerged viruses and other viruses that are not readily culturable. To address these knowledge gaps, this dissertation explores the reactions that occur in viral nucleic acids during photolysis and chlorine disinfection. The research spans several levels of nucleic acid reactivity, from the short nucleic acid oligomer level, up to the entire viral genome incorporated in virus particles. In the first portion of this work, the photochemical reactions that take place in viral RNA oligomers were investigated. Specifically, RNA oligomer segments from the genome of bacteriophage MS2 were exposed to UV254, simulated sunlight, and singlet oxygen (1O2), and the oligomer reaction kinetics were analyzed with RT-qPCR and quantitative MALDI-TOF mass spectrometry (MS). One especially important finding of this work was that quantitative MALDI-TOF-MS detected significantly more RNA modifications than RT-qPCR. This suggests that certain chemical modifications in the RNA are not detected by the reverse transcriptase enzyme. High-resolution ESI-Orbitrap MS identified pyrimidine photohydrates as the major UV254 products, which may have contributed to the discrepancy between the MS- and RT-qPCR-based results. In the second portion, the influence of viral nucleic acid higher order structure on UVC photolysis was examined. We measured the direct UV254 photolysis kinetics of four model viral genomes composed of single-stranded and double-stranded RNA, as well as single-stranded and double-stranded DNA, in ultrapure water, in phosphate buffered saline, and encapsidated in their native virus particles. The photolysis rate constants of naked nucleic acids measured by qPCR (RT-qPCR for RNA) and normalized by the number of bases measured in a particular sequence exhibited the following trend: ssDNA > dsDNA ≈ ssRNA > dsRNA. Interestingly, encapsidation of viral genomes did not affect the photoreactivity of most genome sequences. A large difference in photoreactivity was observed between single and double strands of both RNA and DNA. In the final portion, the impact of viral genome higher order structure on reactivity with free chlorine was characterized. Chlorine reaction kinetics of the same four model viral genomes were measured when they were naked in solution and when they were incorporated in their native virus particles, respectively. We observed that for most of the nucleic acid regions studied, the naked viral genomes reacted with chlorine significantly faster than encapsidated genomes. The research suggests that dsDNA was the least reactive of the genome types tested. Specifically, the two T3 dsDNA regions were ~72 times more resistant than the ssDNA regions, which was the most reactive genome type tested.PHDEnvironmental EngineeringUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttps://deepblue.lib.umich.edu/bitstream/2027.42/147509/1/zhqiao_1.pd

The combined role of NTH and MUTY DNA glycosylases in mycobacterium smegmatis

A dissertation submitted to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Master of Science in Medicine.During infection, Mycobacterium tuberculosis (MTB) encounters hostile conditions such as nutrient starvation, hypoxia and low pH which results in the generation of host-derived reactive oxygen (ROS) and nitrogen species (RNS) as part of the immune response to control the infection. Exposure to these reactive radicals can lead to oxidative damage of DNA which ultimately creates genomic instability through the introduction of mutations. Mycobacterial species have a high G+C content and these bases are particularly prone to oxidative damage. To counter this, MTB possesses specialized DNA repair systems such as the multi-step, multi-enzyme base excision repair (BER) pathway, wherein repair of oxidative damage is initiated by DNA glycosylases. These DNA glycosylases include the endonuclease III (Nth), endonuclease VIII (Nei) and formamidopyrimidine glycosylases (Fpg). In addition, MutY which acts together with Fpg (MutM) and MutT, forms part of the GO system, preventing mutations produced from 7,8-dihydro-8-oxoguanine (8-oxoG) lesions. Previously in our laboratory, we demonstrated a novel antimutator role for Nth and MutY DNA glycosylases in the BER pathway for the maintenance of mycobacterial genome stability. These data showed an increase in spontaneous mutation rate and decreased survival of Mycobacterium smegmatis when the mutY gene was disrupted together with the fpg DNA glycosylases. Additionally, deletion of the nth DNA glycosylase in M. smegmatis resulted in increased mutation rates under DNA damaging conditions and its inactivation in combination with nei resulted in reduced survival in an oxidative environment, with a heightened mutation frequency. In this study, we further investigated the combined role of Nth and MutY in an attempt to uncover the molecular mechanisms that protect mycobacterial DNA under hostile host environments. Double deletion mutants of M. smegmatis mc2155 lacking both the mutY and nth genes (ΔmutYΔnth, ΔnthΔmutY) were generated by homologous recombination. Both mutants displayed no growth defects under normal culture conditions (7H9 media) when compared to the wildtype mc2155 or the respective single M. smegmatis Δnth and ΔmutY mutants. Under in vitro oxidative stress conditions, as generated by hydrogen peroxide, both the double deletion mutants displayed reduced survival kinetics compared to mc2155 after 6 hours of exposure to hydrogen peroxide. As previously observed, loss of the nth gene resulted in increased DNA damage-induced mutation frequencies to rifampicin. In contrast, the mutY single deletion mutant and the double deletion mutants lacking both nth and mutY did not show increased DNA damage-induced mutagenesis compared to mc2155. However, in the fluctuation assay, both double mutant strains, ΔnthΔmutY and ΔmutYΔnth, displayed an increase in spontaneous mutation rates to rifampicin when compared to wild type and the single Δnth/ΔmutY deletion mutants. The ΔnthΔmutY mutant demonstrated an exacerbated mutation rate when compared to the ΔmutYΔnth mutant. Collectively, these data reinforce the previously observed antimutator role for the mycobacterial MutY and Nth DNA glycosylases. The exacerbated phenotype observed for ΔnthΔmutY suggest that there is a functional hierarchy between the various DNA glycosylases in the BER pathway, with the Nth DNA glycosylase superseding MutY as an antimutator in mycobacterial genome maintenance.MT201

UVA Photosensitisers, Protein Oxidation and DNA Repair

Pharmaceuticals can interact with sunlight to cause skin photosensitization and increase skin cancer risk. Interaction of drug molecules with solar UVA or visible radiation results in electronically excited states that damage biomolecules directly or indirectly via the formation of reactive species (RS). RS cause damage to DNA and its precursors, as well as to proteins and lipids. I have devised methods to examine the induction of oxidative protein damage in cultured human cells and used these to investigate the effects of UVA-activated photosensitizing drugs on the formation of protein carbonyls and the oxidation of protein thiol groups. I examined the effects of 6-thioguanine (6-TG) (a surrogate for azathioprine, an immunosuppressant), fluoroquinolone antibiotics, and the malignant melanoma therapeutic vemurafenib, each of which is associated with clinical skin photosensitivity and increased skin cancer risk in patients. All of these drugs are shown to be synergistically cytotoxic with UVA in cultured human cells and toxicity is concurrent with the generation of RS. I identify singlet oxygen as a major component of these photochemically-generated RS and show that widespread protein oxidation is caused. The Ku DNA repair heterodimer is identified as one of several targets for oxidation damage and I show using biochemical assays that damage to Ku compromises its function in the repair of DNA strand breaks. UVA irradiation of cells treated with the photosensitisers significantly compromises the removal of potentially mutagenic DNA lesions by the nucleotide excision repair pathway. Since this DNA repair pathway removes sunlight-induced DNA lesions and is the major protection against skin cancer, my findings have implications for the increased skin cancer risk associated with azathioprine. The ability of structurally dissimilar drugs to recapitulate the effects of 6-TG suggests that the observations may share a common mechanism