A Quantitative Tissue-Specific Landscape of Protein Redox Regulation during Aging.

Mammalian tissues engage in specialized physiology that is regulated through reversible modification of protein cysteine residues by reactive oxygen species (ROS). ROS regulate a myriad of biological processes, but the protein targets of ROS modification that drive tissue-specific physiology in vivo are largely unknown. Here, we develop Oximouse, a comprehensive and quantitative mapping of the mouse cysteine redox proteome in vivo. We use Oximouse to establish several paradigms of physiological redox signaling. We define and validate cysteine redox networks within each tissue that are tissue selective and underlie tissue-specific biology. We describe a common mechanism for encoding cysteine redox sensitivity by electrostatic gating. Moreover, we comprehensively identify redox-modified disease networks that remodel in aged mice, establishing a systemic molecular basis for the long-standing proposed links between redox dysregulation and tissue aging. We provide the Oximouse compendium as a framework for understanding mechanisms of redox regulation in physiology and aging.

Spatial Transcriptomics and In Situ Sequencing to Study Alzheimer's Disease.

Although complex inflammatory-like alterations are observed around the amyloid plaques of Alzheimer's disease (AD), little is known about the molecular changes and cellular interactions that characterize this response. We investigate here, in an AD mouse model, the transcriptional changes occurring in tissue domains in a 100-µm diameter around amyloid plaques using spatial transcriptomics. We demonstrate early alterations in a gene co-expression network enriched for myelin and oligodendrocyte genes (OLIGs), whereas a multicellular gene co-expression network of plaque-induced genes (PIGs) involving the complement system, oxidative stress, lysosomes, and inflammation is prominent in the later phase of the disease. We confirm the majority of the observed alterations at the cellular level using in situ sequencing on mouse and human brain sections. Genome-wide spatial transcriptomics analysis provides an unprecedented approach to untangle the dysregulated cellular network in the vicinity of pathogenic hallmarks of AD and other brain diseases.

Diversity and versatility of p38 kinase signalling in health and disease.

The ability of cells to deal with different types of stressful situations in a precise and coordinated manner is key for survival and involves various signalling networks. Over the past 25 years, p38 kinases - in particular, p38α - have been implicated in the cellular response to stress at many levels. These span from environmental and intracellular stresses, such as hyperosmolarity, oxidative stress or DNA damage, to physiological situations that involve important cellular changes such as differentiation. Given that p38α controls a plethora of functions, dysregulation of this pathway has been linked to diseases such as inflammation, immune disorders or cancer, suggesting the possibility that targeting p38α could be of therapeutic interest. In this Review, we discuss the organization of this signalling pathway focusing on the diversity of p38α substrates, their mechanisms and their links to particular cellular functions. We then address how the different cellular responses can be generated depending on the signal received and the cell type, and highlight the roles of this kinase in human physiology and in pathological contexts.

Cellular adaptation to hypoxia through hypoxia inducible factors and beyond.

Molecular oxygen (O2) sustains intracellular bioenergetics and is consumed by numerous biochemical reactions, making it essential for most species on Earth. Accordingly, decreased oxygen concentration (hypoxia) is a major stressor that generally subverts life of aerobic species and is a prominent feature of pathological states encountered in bacterial infection, inflammation, wounds, cardiovascular defects and cancer. Therefore, key adaptive mechanisms to cope with hypoxia have evolved in mammals. Systemically, these adaptations include increased ventilation, cardiac output, blood vessel growth and circulating red blood cell numbers. On a cellular level, ATP-consuming reactions are suppressed, and metabolism is altered until oxygen homeostasis is restored. A critical question is how mammalian cells sense oxygen levels to coordinate diverse biological outputs during hypoxia. The best-studied mechanism of response to hypoxia involves hypoxia inducible factors (HIFs), which are stabilized by low oxygen availability and control the expression of a multitude of genes, including those involved in cell survival, angiogenesis, glycolysis and invasion/metastasis. Importantly, changes in oxygen can also be sensed via other stress pathways as well as changes in metabolite levels and the generation of reactive oxygen species by mitochondria. Collectively, this leads to cellular adaptations of protein synthesis, energy metabolism, mitochondrial respiration, lipid and carbon metabolism as well as nutrient acquisition. These mechanisms are integral inputs into fine-tuning the responses to hypoxic stress.

ALOX5-mediated ferroptosis acts as a distinct cell death pathway upon oxidative stress in Huntington's disease.

Although it is well established that Huntington's disease (HD) is mainly caused by polyglutamine-expanded mutant huntingtin (mHTT), the molecular mechanism of mHTT-mediated actions is not fully understood. Here, we showed that expression of the N-terminal fragment containing the expanded polyglutamine (HTTQ94) of mHTT is able to promote both the ACSL4-dependent and the ACSL4-independent ferroptosis. Surprisingly, inactivation of the ACSL4-dependent ferroptosis fails to show any effect on the life span of Huntington's disease mice. Moreover, by using RNAi-mediated screening, we identified ALOX5 as a major factor required for the ACSL4-independent ferroptosis induced by HTTQ94. Although ALOX5 is not required for the ferroptotic responses triggered by common ferroptosis inducers such as erastin, loss of ALOX5 expression abolishes HTTQ94-mediated ferroptosis upon reactive oxygen species (ROS)-induced stress. Interestingly, ALOX5 is also required for HTTQ94-mediated ferroptosis in neuronal cells upon high levels of glutamate. Mechanistically, HTTQ94 activates ALOX5-mediated ferroptosis by stabilizing FLAP, an essential cofactor of ALOX5-mediated lipoxygenase activity. Notably, inactivation of the Alox5 gene abrogates the ferroptosis activity in the striatal neurons from the HD mice; more importantly, loss of ALOX5 significantly ameliorates the pathological phenotypes and extends the life spans of these HD mice. Taken together, these results demonstrate that ALOX5 is critical for mHTT-mediated ferroptosis and suggest that ALOX5 is a potential new target for Huntington's disease.

A mechanism for oxidative damage repair at gene regulatory elements.

Oxidative genome damage is an unavoidable consequence of cellular metabolism. It arises at gene regulatory elements by epigenetic demethylation during transcriptional activation1,2. Here we show that promoters are protected from oxidative damage via a process mediated by the nuclear mitotic apparatus protein NuMA (also known as NUMA1). NuMA exhibits genomic occupancy approximately 100 bp around transcription start sites. It binds the initiating form of RNA polymerase II, pause-release factors and single-strand break repair (SSBR) components such as TDP1. The binding is increased on chromatin following oxidative damage, and TDP1 enrichment at damaged chromatin is facilitated by NuMA. Depletion of NuMA increases oxidative damage at promoters. NuMA promotes transcription by limiting the polyADP-ribosylation of RNA polymerase II, increasing its availability and release from pausing at promoters. Metabolic labelling of nascent RNA identifies genes that depend on NuMA for transcription including immediate-early response genes. Complementation of NuMA-deficient cells with a mutant that mediates binding to SSBR, or a mitotic separation-of-function mutant, restores SSBR defects. These findings underscore the importance of oxidative DNA damage repair at gene regulatory elements and describe a process that fulfils this function.

Autophagy promotes mammalian survival by suppressing oxidative stress and p53.

Autophagy captures intracellular components and delivers them to lysosomes for degradation and recycling. Conditional autophagy deficiency in adult mice causes liver damage, shortens life span to 3 mo due to neurodegeneration, and is lethal upon fasting. As autophagy deficiency causes p53 induction and cell death in neurons, we sought to test whether p53 mediates the lethal consequences of autophagy deficiency. Here, we conditionally deleted Trp53 (p53 hereafter) and/or the essential autophagy gene Atg7 throughout adult mice. Compared with Atg7Δ/Δ mice, the life span of Atg7Δ/Δp53Δ/Δ mice was extended due to delayed neurodegeneration and resistance to death upon fasting. Atg7 also suppressed apoptosis induced by p53 activator Nutlin-3, suggesting that autophagy inhibited p53 activation. To test whether increased oxidative stress in Atg7Δ/Δ mice was responsible for p53 activation, Atg7 was deleted in the presence or absence of the master regulator of antioxidant defense nuclear factor erythroid 2-related factor 2 (Nrf2). Nrf2-/-Atg7Δ/Δ mice died rapidly due to small intestine damage, which was not rescued by p53 codeletion. Thus, Atg7 limits p53 activation and p53-mediated neurodegeneration. In turn, NRF2 mitigates lethal intestine degeneration upon autophagy loss. These findings illustrate the tissue-specific roles for autophagy and functional dependencies on the p53 and NRF2 stress response mechanisms.

Oxidative stress accelerates intestinal tumorigenesis by enhancing 8-oxoguanine-mediated mutagenesis in MUTYH-deficient mice.

Oxidative stress-induced DNA damage and its repair systems are related to cancer etiology; however, the molecular basis triggering tumorigenesis is not well understood. Here, we aimed to explore the causal relationship between oxidative stress, somatic mutations in pre-tumor-initiated normal tissues, and tumor incidence in the small intestines of MUTYH-proficient and MUTYH-deficient mice. MUTYH is a base excision repair enzyme associated with human colorectal cancer. Mice were administered different concentrations of potassium bromate (KBrO3; an oxidizing agent)-containing water for 4 wk for mutagenesis studies or 16 wk for tumorigenesis studies. All Mutyh -/- mice treated with >0.1% KBrO3 developed multiple tumors, and the average tumor number increased dose dependently. Somatic mutation analysis of Mutyh -/-/rpsL transgenic mice revealed that G:C  > T:A transversion was the only mutation type correlated positively with KBrO3 dose and tumor incidence. These mutations preferentially occurred at 5'G in GG and GAA sequences in rpsL This characteristic mutation pattern was also observed in the genomic region of Mutyh -/- tumors using whole-exome sequencing. It closely corresponded to signature 18 and SBS36, typically caused by 8-oxo-guanine (8-oxoG). 8-oxoG-induced mutations were sequence context dependent, yielding a biased amino acid change leading to missense and stop-gain mutations. These mutations frequently occurred in critical amino acid codons of known cancer drivers, Apc or Ctnnb1, known for activating Wnt signal pathway. Our results indicate that oxidative stress contributes to increased tumor incidence by elevating the likelihood of gaining driver mutations by increasing 8-oxoG-mediated mutagenesis, particularly under MUTYH-deficient conditions.

MDM2 Integrates Cellular Respiration and Apoptotic Signaling through NDUFS1 and the Mitochondrial Network.

Signaling diversity and subsequent complexity in higher eukaryotes is partially explained by one gene encoding a polypeptide with multiple biochemical functions in different cellular contexts. For example, mouse double minute 2 (MDM2) is functionally characterized as both an oncogene and a tumor suppressor, yet this dual classification confounds the cell biology and clinical literatures. Identified via complementary biochemical, organellar, and cellular approaches, we report that MDM2 negatively regulates NADH:ubiquinone oxidoreductase 75 kDa Fe-S protein 1 (NDUFS1), leading to decreased mitochondrial respiration, marked oxidative stress, and commitment to the mitochondrial pathway of apoptosis. MDM2 directly binds and sequesters NDUFS1, preventing its mitochondrial localization and ultimately causing complex I and supercomplex destabilization and inefficiency of oxidative phosphorylation. The MDM2 amino-terminal region is sufficient to bind NDUFS1, alter supercomplex assembly, and induce apoptosis. Finally, this pathway is independent of p53, and several mitochondrial phenotypes are observed in Drosophila and murine models expressing transgenic Mdm2.

Fluorescent proteins generate a genetic color polymorphism and counteract oxidative stress in intertidal sea anemones.

Fluorescent proteins (FPs) are ubiquitous tools in research, yet their endogenous functions in nature are poorly understood. In this work, we describe a combination of functions for FPs in a clade of intertidal sea anemones whose FPs control a genetic color polymorphism together with the ability to combat oxidative stress. Focusing on the underlying genetics of a fluorescent green "Neon" color morph, we show that allelic differences in a single FP gene generate its strong and vibrant color, by increasing both molecular brightness and FP gene expression level. Natural variation in FP sequences also produces differences in antioxidant capacity. We demonstrate that these FPs are strong antioxidants that can protect live cells against oxidative stress. Finally, based on structural modeling of the responsible amino acids, we propose a model for FP antioxidant function that is driven by molecular surface charge. Together, our findings shed light on the multifaceted functions that can co-occur within a single FP and provide a framework for studying the evolution of fluorescence as it balances spectral and physiological functions in nature.

Unraveling the genetics of arsenic toxicity with cellular morphology QTL.

The health risks that arise from environmental exposures vary widely within and across human populations, and these differences are largely determined by genetic variation and gene-by-environment (gene-environment) interactions. However, risk assessment in laboratory mice typically involves isogenic strains and therefore, does not account for these known genetic effects. In this context, genetically heterogenous cell lines from laboratory mice are promising tools for population-based screening because they provide a way to introduce genetic variation in risk assessment without increasing animal use. Cell lines from genetic reference populations of laboratory mice offer genetic diversity, power for genetic mapping, and potentially, predictive value for in vivo experimentation in genetically matched individuals. To explore this further, we derived a panel of fibroblast lines from a genetic reference population of laboratory mice (the Diversity Outbred, DO). We then used high-content imaging to capture hundreds of cell morphology traits in cells exposed to the oxidative stress-inducing arsenic metabolite monomethylarsonous acid (MMAIII). We employed dose-response modeling to capture latent parameters of response and we then used these parameters to identify several hundred cell morphology quantitative trait loci (cmQTL). Response cmQTL encompass genes with established associations with cellular responses to arsenic exposure, including Abcc4 and Txnrd1, as well as novel gene candidates like Xrcc2. Moreover, baseline trait cmQTL highlight the influence of natural variation on fundamental aspects of nuclear morphology. We show that the natural variants influencing response include both coding and non-coding variation, and that cmQTL haplotypes can be used to predict response in orthogonal cell lines. Our study sheds light on the major molecular initiating events of oxidative stress that are under genetic regulation, including the NRF2-mediated antioxidant response, cellular detoxification pathways, DNA damage repair response, and cell death trajectories.

Sequestrase chaperones protect against oxidative stress-induced protein aggregation and [PSI+] prion formation.

Misfolded proteins are usually refolded to their functional conformations or degraded by quality control mechanisms. When misfolded proteins evade quality control, they can be sequestered to specific sites within cells to prevent the potential dysfunction and toxicity that arises from protein aggregation. Btn2 and Hsp42 are compartment-specific sequestrases that play key roles in the assembly of these deposition sites. Their exact intracellular functions and substrates are not well defined, particularly since heat stress sensitivity is not observed in deletion mutants. We show here that Btn2 and Hsp42 are required for tolerance to oxidative stress conditions induced by exposure to hydrogen peroxide. Btn2 and Hsp42 act to sequester oxidized proteins into defined PQC sites following ROS exposure and their absence leads to an accumulation of protein aggregates. The toxicity of protein aggregate accumulation causes oxidant sensitivity in btn2 hsp42 sequestrase mutants since overexpression of the Hsp104 disaggregase rescues oxidant tolerance. We have identified the Sup35 translation termination factor as an in vivo sequestrase substrate and show that Btn2 and Hsp42 act to suppress oxidant-induced formation of the yeast [PSI+] prion, which is the amyloid form of Sup35. [PSI+] prion formation in sequestrase mutants does not require IPOD (insoluble protein deposit) localization which is the site where amyloids are thought to undergo fragmentation and seeding to propagate their heritable prion form. Instead, both amorphous and amyloid Sup35 aggregates are increased in btn2 hsp42 mutants consistent with the idea that prion formation occurs at multiple intracellular sites during oxidative stress conditions in the absence of sequestrase activity. Taken together, our data identify protein sequestration as a key antioxidant defence mechanism that functions to mitigate the damaging consequences of protein oxidation-induced aggregation.

snRNA-seq analysis in multinucleated myogenic FSHD cells identifies heterogeneous FSHD transcriptome signatures associated with embryonic-like program activation and oxidative stress-induced apoptosis.

The sporadic nature of DUX4 expression in FSHD muscle challenges comparative transcriptome analyses between FSHD and control samples. A variety of DUX4 and FSHD-associated transcriptional changes have been identified, but bulk RNA-seq strategies prohibit comprehensive analysis of their spatiotemporal relation, interdependence and role in the disease process. In this study, we used single-nucleus RNA-sequencing of nuclei isolated from patient- and control-derived multinucleated primary myotubes to investigate the cellular heterogeneity in FSHD. Taking advantage of the increased resolution in snRNA-sequencing of fully differentiated myotubes, two distinct populations of DUX4-affected nuclei could be defined by their transcriptional profiles. Our data provides insights into the differences between these two populations and suggests heterogeneity in two well-known FSHD-associated transcriptional aberrations: increased oxidative stress and inhibition of myogenic differentiation. Additionally, we provide evidence that DUX4-affected nuclei share transcriptome features with early embryonic cells beyond the well-described cleavage stage, progressing into the 8-cell and blastocyst stages. Altogether, our data suggests that the FSHD transcriptional profile is defined by a mixture of individual and sometimes mutually exclusive DUX4-induced responses and cellular state-dependent downstream effects.

Clinical mutations in the TERT and TERC genes coding for telomerase components induced oxidative stress, DNA damage at telomeres and cell apoptosis besides decreased telomerase activity.

Telomeres are nucleoprotein structures at the end of chromosomes that maintain their integrity. Mutations in genes coding for proteins involved in telomere protection and elongation produce diseases such as dyskeratosis congenita or idiopathic pulmonary fibrosis known as telomeropathies. These diseases are characterized by premature telomere shortening, increased DNA damage and oxidative stress. Genetic diagnosis of telomeropathy patients has identified mutations in the genes TERT and TERC coding for telomerase components but the functional consequences of many of these mutations still have to be experimentally demonstrated. The activity of twelve TERT and five TERC mutants, five of them identified in Spanish patients, has been analyzed. TERT and TERC mutants were expressed in VA-13 human cells that express low telomerase levels and the activity induced was analyzed. The production of reactive oxygen species, DNA oxidation and TRF2 association at telomeres, DNA damage response and cell apoptosis were determined. Most mutations presented decreased telomerase activity, as compared to wild-type TERT and TERC. In addition, the expression of several TERT and TERC mutants induced oxidative stress, DNA oxidation, DNA damage, decreased recruitment of the shelterin component TRF2 to telomeres and increased apoptosis. These observations might indicate that the increase in DNA damage and oxidative stress observed in cells from telomeropathy patients is dependent on their TERT or TERC mutations. Therefore, analysis of the effect of TERT and TERC mutations of unknown function on DNA damage and oxidative stress could be of great utility to determine the possible pathogenicity of these variants.

Pathways controlling neurotoxicity and proteostasis in mitochondrial complex I deficiency.

Neuromuscular disorders caused by dysfunction of the mitochondrial respiratory chain are common, severe and untreatable. We recovered a number of mitochondrial genes, including electron transport chain components, in a large forward genetic screen for mutations causing age-related neurodegeneration in the context of proteostasis dysfunction. We created a model of complex I deficiency in the Drosophila retina to probe the role of protein degradation abnormalities in mitochondrial encephalomyopathies. Using our genetic model, we found that complex I deficiency regulates both the ubiquitin/proteasome and autophagy/lysosome arms of the proteostasis machinery. We further performed an in vivo kinome screen to uncover new and potentially druggable mechanisms contributing to complex I related neurodegeneration and proteostasis failure. Reduction of RIOK kinases and the innate immune signaling kinase pelle prevented neurodegeneration in complex I deficiency animals. Genetically targeting oxidative stress, but not RIOK1 or pelle knockdown, normalized proteostasis markers. Our findings outline distinct pathways controlling neurodegeneration and protein degradation in complex I deficiency and introduce an experimentally facile model in which to study these debilitating and currently treatment-refractory disorders.

Gre factors help Salmonella adapt to oxidative stress by improving transcription elongation and fidelity of metabolic genes.

Detoxification, scavenging, and repair systems embody the archetypical antioxidant defenses of prokaryotic and eukaryotic cells. Metabolic rewiring also aids with the adaptation of bacteria to oxidative stress. Evolutionarily diverse bacteria combat the toxicity of reactive oxygen species (ROS) by actively engaging the stringent response, a stress program that controls many metabolic pathways at the level of transcription initiation via guanosine tetraphosphate and the α-helical DksA protein. Studies herein with Salmonella demonstrate that the interactions of structurally related, but functionally unique, α-helical Gre factors with the secondary channel of RNA polymerase elicit the expression of metabolic signatures that are associated with resistance to oxidative killing. Gre proteins both improve transcriptional fidelity of metabolic genes and resolve pauses in ternary elongation complexes of Embden-Meyerhof-Parnas (EMP) glycolysis and aerobic respiration genes. The Gre-directed utilization of glucose in overflow and aerobic metabolism satisfies the energetic and redox demands of Salmonella, while preventing the occurrence of amino acid bradytrophies. The resolution of transcriptional pauses in EMP glycolysis and aerobic respiration genes by Gre factors safeguards Salmonella from the cytotoxicity of phagocyte NADPH oxidase in the innate host response. In particular, the activation of cytochrome bd protects Salmonella from phagocyte NADPH oxidase-dependent killing by promoting glucose utilization, redox balancing, and energy production. Control of transcription fidelity and elongation by Gre factors represent important points in the regulation of metabolic programs supporting bacterial pathogenesis.

Leishmania allelic selection during experimental sand fly infection correlates with mutational signatures of oxidative DNA damage.

Trypanosomatid pathogens are transmitted by blood-feeding insects, causing devastating human infections. These parasites show important phenotypic shifts that often impact parasite pathogenicity, tissue tropism, or drug susceptibility. The evolutionary mechanisms that allow for the selection of such adaptive phenotypes remain only poorly investigated. Here, we use Leishmania donovani as a trypanosomatid model pathogen to assess parasite evolutionary adaptation during experimental sand fly infection. Comparing the genome of the parasites before and after sand fly infection revealed a strong population bottleneck effect as judged by allele frequency analysis. Apart from random genetic drift caused by the bottleneck effect, our analyses revealed haplotype and allelic changes during sand fly infection that seem under natural selection given their convergence between independent biological replicates. Our analyses further uncovered signature mutations of oxidative DNA damage in the parasite genomes after sand fly infection, suggesting that Leishmania suffers from oxidative stress inside the insect digestive tract. Our results propose a model of Leishmania genomic adaptation during sand fly infection, with oxidative DNA damage and DNA repair processes likely driving haplotype and allelic selection. The experimental and computational framework presented here provides a useful blueprint to assess evolutionary adaptation of other eukaryotic pathogens inside their insect vectors, such as Plasmodium spp, Trypanosoma brucei, and Trypanosoma cruzi.

Sensing of H2O2-induced oxidative stress by the UPF factor complex is crucial for activation of catalase-3 expression in Neurospora.

UPF-1-UPF-2-UPF-3 complex-orchestrated nonsense-mediated mRNA decay (NMD) is a well-characterized eukaryotic cellular surveillance mechanism that not only degrades aberrant transcripts to protect the integrity of the transcriptome but also eliminates normal transcripts to facilitate appropriate cellular responses to physiological and environmental changes. Here, we describe the multifaceted regulatory roles of the Neurospora crassa UPF complex in catalase-3 (cat-3) gene expression, which is essential for scavenging H2O2-induced oxidative stress. First, losing UPF proteins markedly slowed down the decay rate of cat-3 mRNA. Second, UPF proteins indirectly attenuated the transcriptional activity of cat-3 gene by boosting the decay of cpc-1 and ngf-1 mRNAs, which encode a well-studied transcription factor and a histone acetyltransferase, respectively. Further study showed that under oxidative stress condition, UPF proteins were degraded, followed by increased CPC-1 and NGF-1 activity, finally activating cat-3 expression to resist oxidative stress. Together, our data illustrate a sophisticated regulatory network of the cat-3 gene mediated by the UPF complex under physiological and H2O2-induced oxidative stress conditions.

Oxidative stress drives mutagenesis through transcription-coupled repair in bacteria.

In bacteria, mutations lead to the evolution of antibiotic resistance, which is one of the main public health problems of the twenty-first century. Therefore, determining which cellular processes most frequently contribute to mutagenesis, especially in cells that have not been exposed to exogenous DNA damage, is critical. Here, we show that endogenous oxidative stress is a key driver of mutagenesis and the subsequent development of antibiotic resistance. This is the case for all classes of antibiotics and highly divergent species tested, including patient-derived strains. We show that the transcription-coupled repair pathway, which uses the nucleotide excision repair proteins (TC-NER), is responsible for endogenous oxidative stress-dependent mutagenesis and subsequent evolution. This suggests that a majority of mutations arise through transcription-associated processes rather than the replication fork. In addition to determining that the NER proteins play a critical role in mutagenesis and evolution, we also identify the DNA polymerases responsible for this process. Our data strongly suggest that cooperation between three different mutagenic DNA polymerases, likely at the last step of TC-NER, is responsible for mutagenesis and evolution. Overall, our work identifies a highly conserved pathway that drives mutagenesis due to endogenous oxidative stress, which has broad implications for all diseases of evolution, including antibiotic resistance development.

8-oxoguanine riboswitches in bacteria detect and respond to oxidative DNA damage.

Riboswitches rely on structured aptamer domains to selectively sense their target ligands and regulate gene expression. However, some riboswitch aptamers in bacteria carry mutations in their otherwise strictly conserved binding pockets that change ligand specificities. The aptamer domain of a riboswitch class originally found to selectively sense guanine forms a three-stem junction that has since been observed to exploit numerous alterations in its ligand-binding pocket. These rare variants have modified their ligand specificities to sense other purines or purine derivatives, including adenine, 2'-deoxyguanosine (three classes), and xanthine. Herein, we report the characteristics of a rare variant that is narrowly distributed in the Paenibacillaceae family of bacteria. Known representatives are always associated with genes encoding 8-oxoguanine deaminase. As predicted from this gene association, these variant riboswitches tightly bind 8-oxoguanine (8-oxoG), strongly discriminate against other purine derivatives, and function as genetic "ON" switches. Following exposure of cells to certain oxidative stresses, a representative 8-oxoG riboswitch activates gene expression, likely caused by the accumulation of 8-oxoG due to oxidative damage to G nucleobases in DNA, RNA, and the nucleotide pool. Furthermore, an engineered version of the variant aptamer was prepared that exhibits specificity for 8-oxoadenine, further demonstrating that RNA aptamers can acquire mutations that expand their ability to detect and respond to oxidative damage.