Ribonucleotide incorporation into mitochondrial DNA induces cGAS-STING dependent inflammation
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ABSTRACT:
PROVIDER: PRJEB87869 | ENA |
REPOSITORIES: ENA
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Project description:All DNA polymerases misincorporate ribonucleotides despite their preference for deoxyribonucleotides, and analysis of cultured cells indicates that mammalian mitochondrial DNA (mtDNA) tolerates such replication errors. However, it is not clear to what extent ribonucleotides are incorporated into the mtDNA of solid tissues, or whether they might play a role in human pathologies. Here, we show the DNA in mitochondria of solid tissues contains many more embedded ribonucleotides than that of cultured cells, consistent with the former’s high ratio of ribonucleotide to deoxynucleotide triphosphates and that rAMPs are the predominant ribosubstitution events. This pattern changes in a mouse model of Mpv17 deficiency, as rGMPs are the major embedded ribonucleotides of mtDNA. However, while mitochondrial dGTP is reduced in the liver of the KO mice, the brain shows no change in the overall dGTP pool, leading us to infer that Mpv17 determines the local concentration or quality of dGTP. Embedded rGMPs are expected to impede DNA replication more than other rNMPs, and elevated rGMP incorporation is associated with early-onset mtDNA depletion in liver and late-onset multiple deletions in brain of the Mpv17 ablated mice. These findings suggest that aberrant ribonucleotide incorporation is a primary mtDNA abnormality that can result in pathology.
2017-10-23 | GSE103429 | GEO
Project description:Previous work has demonstrated the presence of ribonucleotides in human mitochondrial DNA (mtDNA) and in the present study we use a genome-wide approach to precisely map the location of these. We find that ribonucleotides are distributed evenly between the heavy- and light-strand of mtDNA. The relative levels of incorporated ribonucleotides reflect that DNA polymerase γ discriminates the four ribonucleotides differentially during DNA synthesis. The observed pattern is also dependent on the mitochondrial deoxyribonucleotide (dNTP) pools and disease-causing mutations that change these pools alter both the absolute and relative levels of incorporated ribonucleotides. Our analyses strongly suggest that DNA polymerase γ-dependent incorporation is the main source of ribonucleotides in mtDNA and argues against the existence of a mitochondrial ribonucleotide excision repair pathway in human cells. Furthermore, we clearly demonstrate that when dNTP pools are limiting, ribonucleotides serve as a source of building blocks to maintain DNA replication and genome stability. Increased levels of embedded ribonucleotides in patient cells with disturbed nucleotide pools may constitute to a pathogenic mechanism that affects mtDNA stability and impair new rounds of mtDNA replication
2017-02-13 | GSE90054 | GEO
Project description:We devised and improved on our hydrolytic end sequencing (HydEn-seq) that mapping ribonucleotide incorporation in genome and used this method to track DNA replicative polymerase usage. We uncovered striking exceptions to canonical polymerase division of labor in Saccharomyces cerevisiae and Schizosaccharomyces pombe.
2019-01-30 | GSE125855 | GEO
Project description:Multiple DNA polymerases are needed to replicate genetic information. Here we describe the use of ribonucleotide incorporation as a biomarker of replication enzymology in vivo. We find that ribonucleotides are incorporated into the yeast nuclear genome in replicase specific and strand-specific patterns that identify replication origins and where polymerase switching occurs. Ribonucleotide density varies across the genome as a function of the replicase, base, local sequence and proximity to nucleosomes and transcription start sites. Ribonucleotides are present in one strand at high densityat mitochondrial replication origins, implying unidirectional replication of a circular genome. The evolutionary conservation of the enzymes that incorporate and process ribonucleotides in DNA suggests that the use of ribonucleotides as biomarkers of DNA synthesis in cells will have widespread applicability. Mapping genomic ribonucleotides in 14 Saccharomyces cerevisiae strains (seven DNA polymerase backgrounds, with or without RNH201), via HydEn-seq (end sequencing of genomic fragments generated by alkaline hydrolysis).
2015-01-26 | E-GEOD-62181 | biostudies-arrayexpress
Project description:Multiple DNA polymerases are needed to replicate genetic information. Here we describe the use of ribonucleotide incorporation as a biomarker of replication enzymology in vivo. We find that ribonucleotides are incorporated into the yeast nuclear genome in replicase specific and strand-specific patterns that identify replication origins and where polymerase switching occurs. Ribonucleotide density varies across the genome as a function of the replicase, base, local sequence and proximity to nucleosomes and transcription start sites. Ribonucleotides are present in one strand at high densityat mitochondrial replication origins, implying unidirectional replication of a circular genome. The evolutionary conservation of the enzymes that incorporate and process ribonucleotides in DNA suggests that the use of ribonucleotides as biomarkers of DNA synthesis in cells will have widespread applicability.
2015-01-26 | GSE62181 | GEO
Project description:Aberrant ribonucleotide incorporation and multiple deletions in mitochondrial DNA of the murine MPV17 disease model
| PRJEB23087 | ENA
Project description:Mapping polymerase usage by tracking ribonucleotide incorporation (HydEn-seq) during DNA replication
| PRJNA517710 | ENA
Project description:The innate immune response is the main pathophysiological process of ocular surface diseases exposed to multiple environmental stresses. The epithelium is central to the innate immune response, but whether and how innate immunity is initiated by ocular epithelial cells in response to various environmental stresses in ocular surface diseases, such as dry eye, is still unclear. By utilizing two classic experimental dry eye models-a mouse ocular surface treated with benzalkonium chloride (BAC) and a mouse model with surgically removed extraorbital lachrymal glands, as well as dry eye patient samples-along with human corneal epithelial cells (HCE) exposed to hyperosmolarity, we have discovered a novel innate immune pathway in ocular surface epithelial cells. Under stress, mitochondrial DNA (mtDNA) was released into the cytoplasm through the mitochondrial permeability transition pore (mPTP) and further activated the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway, aggravating downstream inflammatory responses and ocular surface damage. Genetic deletion or pharmacological suppression of STING and inhibition of mtDNA release reduced inflammatory responses, whereas mtDNA transfection supported cytoplasmic mtDNA-induced inflammatory responses by activating the cGAS-STING pathway. Our study clarified the cGAS-STING pathway-dependent sensing of mitochondrial DNA-mediated ocular surface inflammation, which elucidated a new mechanism of ocular surface diseases in response to multiple environmental stresses.
| S-EPMC10514335 | biostudies-literature
Project description:Metabolic dysregulation can lead to inflammatory responses. Imbalanced nucleotide synthesis triggers the release of mitochondrial DNA (mtDNA) to the cytosol and an innate immune response by cGAS-STING signaling. However, how nucleotide deficiency drives mtDNA dependent inflammation has not been elucidated. Here, we show that nucleotide imbalance leads to an increased misincorporation of ribonucleotides into mtDNA during age-dependent renal inflammation in a mouse model lacking the mitochondrial exonuclease MGME1 and upon inhibition of pyrimidine synthesis in cells lacking the mitochondrial i-AAA protease YME1L. Similarly, reduced deoxyribonucleotide synthesis increases ribonucleotide content of mtDNA in cell cycle-arrested senescent cells. This leads to mtDNA release into the cytosol, cGAS-STING activation and the mtDNA-dependent senescence-associated secretory phenotype (SASP), which can be suppressed by exogenously added deoxyribonucleotides. Our results explain age- and mtDNA-dependent inflammatory responses by imbalanced nucleotide metabolism and highlight the sensitivity of mtDNA to aberrant ribonucleotide incorporation.
2025-07-25 | PXD053639 | Pride
Project description:Antimetabolite chemotherapies increase uracil levels in DNA, and thus identification of factors that influence the uracil content in DNA may have implications for understanding uracil-mediated chromosomal instability. We previously showed in the budding yeast Saccharomyces cerevisiae that uracil content in DNA correlates with replication timing, where the earliest and latest replicating regions are depleted in uracil. Here, we manipulated nucleotide biosynthesis enzymes in budding yeast to determine whether the pattern of uracil incorporation could be altered. In strains with high levels of uracil incorporation, deletion of dCMP deaminase (Dcd1) accelerated uracil incorporation at early-firing origins, likely due to rapid dTTP pool depletion. In contrast, increasing the activity of ribonucleotide reductase, which is required for the synthesis of all dNTPs via ribonucleotide diphosphates, lead to dUTP and dTTP pool equilibration and a concomitant increase in uracil content throughout the genome. These data suggest that uracil availability and the dUTP:dTTP ratio are temporally regulated during S phase and govern uracil incorporation into the genome. Therapeutic manipulation of nucleotide biosynthesis in human cells to either increase the dUTP pool or deplete the dTTP pool in early S phase may therefore improve the efficacy of antimetabolite chemotherapies.
2016-09-23 | GSE73364 | GEO