SUMOylation of yeast Pso2 enhances its translocation and accumulation in the mitochondria and suppresses methyl methanesulfonate-induced mitochondrial DNA damage.

Saccharomyces cerevisiae Pso2/SNM1 is essential for DNA interstrand crosslink (ICL) repair; however, its mechanism of action remains incompletely understood. While recent work has revealed that Pso2/Snm1 is dual-localized in the nucleus and mitochondria, it remains unclear whether cell-intrinsic and -extrinsic factors regulate its subcellular localization and function. Herein, we show that Pso2 undergoes ubiquitination and phosphorylation, but not SUMOylation, in unstressed cells. Unexpectedly, we found that methyl methanesulfonate (MMS), rather than ICL-forming agents, induced robust SUMOylation of Pso2 on two conserved residues, K97 and K575, and that SUMOylation markedly increased its abundance in the mitochondria. Reciprocally, SUMOylation had no discernible impact on Pso2 translocation to the nucleus, despite the presence of steady-state levels of SUMOylated Pso2 across the cell cycle. Furthermore, substitution of the invariant residues K97 and K575 by arginine in the Pso2 SUMO consensus motifs severely impaired SUMOylation and abolished its translocation to the mitochondria of MMS-treated wild type cells, but not in unstressed cells. We demonstrate that whilst Siz1 and Siz2 SUMO E3 ligases catalyze Pso2 SUMOylation, the former plays a dominant role. Notably, we found that the phenotypic characteristics of the SUMOylation-defective mutant Pso2K97R/K575R closely mirrored those observed in the Pso2Δ petite mutant. Additionally, leveraging next-generation sequencing analysis, we demonstrate that Pso2 mitigates MMS-induced damage to mitochondrial DNA (mtDNA). Viewed together, our work offers previously unknown insights into the link between genotoxic stress-induced SUMOylation of Pso2 and its preferential targeting to the mitochondria, as well as its role in attenuating MMS-induced mtDNA damage.

Biallelic variants in LIG3 cause a novel mitochondrial neurogastrointestinal encephalomyopathy.

Abnormal gut motility is a feature of several mitochondrial encephalomyopathies, and mutations in genes such as TYMP and POLG, have been linked to these rare diseases. The human genome encodes three DNA ligases, of which only one, ligase III (LIG3), has a mitochondrial splice variant and is crucial for mitochondrial health. We investigated the effect of reduced LIG3 activity and resulting mitochondrial dysfunction in seven patients from three independent families, who showed the common occurrence of gut dysmotility and neurological manifestations reminiscent of mitochondrial neurogastrointestinal encephalomyopathy. DNA from these patients was subjected to whole exome sequencing. In all patients, compound heterozygous variants in a new disease gene, LIG3, were identified. All variants were predicted to have a damaging effect on the protein. The LIG3 gene encodes the only mitochondrial DNA (mtDNA) ligase and therefore plays a pivotal role in mtDNA repair and replication. In vitro assays in patient-derived cells showed a decrease in LIG3 protein levels and ligase activity. We demonstrated that the LIG3 gene defects affect mtDNA maintenance, leading to mtDNA depletion without the accumulation of multiple deletions as observed in other mitochondrial disorders. This mitochondrial dysfunction is likely to cause the phenotypes observed in these patients. The most prominent and consistent clinical signs were severe gut dysmotility and neurological abnormalities, including leukoencephalopathy, epilepsy, migraine, stroke-like episodes, and neurogenic bladder. A decrease in the number of myenteric neurons, and increased fibrosis and elastin levels were the most prominent changes in the gut. Cytochrome c oxidase (COX) deficient fibres in skeletal muscle were also observed. Disruption of lig3 in zebrafish reproduced the brain alterations and impaired gut transit in vivo. In conclusion, we identified variants in the LIG3 gene that result in a mitochondrial disease characterized by predominant gut dysmotility, encephalopathy, and neuromuscular abnormalities.

Deletion of OGG1 Results in a Differential Signature of Oxidized Purine Base Damage in mtDNA Regions.

Mitochondrial oxidative stress accumulates with aging and age-related diseases and induces alterations in mitochondrial DNA (mtDNA) content. Since mtDNA qualitative alterations are also associated with aging, repair of mtDNA damage is of great importance. The most relevant form of DNA repair in this context is base excision repair (BER), which removes oxidized bases such as 8-oxoguanine (8-oxoG) and thymine glycol through the action of the mitochondrial isoform of the specific 8-oxoG DNA glycosylase/apurinic or apyrimidinic (AP) lyase (OGG1) or the endonuclease III homolog (NTH1). Mouse strains lacking OGG1 (OGG1-/-) or NTH1 (NTH1-/-) were analyzed for mtDNA alterations. Interestingly, both knockout strains presented a significant increase in mtDNA content, suggestive of a compensatory mtDNA replication. The mtDNA "common deletion" was not detected in either knockout mouse strain, likely because of the young age of the mice. Formamidopyrimidine DNA glycosylase (Fpg)-sensitive sites accumulated in mtDNA from OGG1-/- but not from NTH1-/- mice. Interestingly, the D-loop region was most severely affected by the absence of OGG1, suggesting that this region may be a hotspot for oxidative damage. Thus, we speculate that mtDNA alterations may send a stress message to evoke cell changes through a retrograde mitochondrial-nucleus communication.

Mitochondrial DNA repair: a novel therapeutic target for heart failure.

Mitochondria play a crucial role in a variety of cellular processes ranging from energy metabolism, generation of reactive oxygen species (ROS) and Ca(2+) handling to stress responses, cell survival and death. Malfunction of the organelle may contribute to the pathogenesis of neuromuscular, cancer, premature aging and cardiovascular diseases (CVD), including myocardial ischemia, cardiomyopathy and heart failure (HF). Mitochondria contain their own genome organized into DNA-protein complexes, called "mitochondrial nucleoids," along with multiprotein machineries, which promote mitochondrial DNA (mtDNA) replication, transcription and repair. Although the mammalian organelle possesses almost all known nuclear DNA repair pathways, including base excision repair, mismatch repair and recombinational repair, the proximity of mtDNA to the main sites of ROS production and the lack of protective histones may result in increased susceptibility to various types of mtDNA damage. These include accumulation of mtDNA point mutations and/or deletions and decreased mtDNA copy number, which will impair mitochondrial function and finally, may lead to CVD including HF.

Mitochondrial DNA maintenance: an appraisal.

Mitochondria play a crucial role in a variety of cellular processes ranging from energy metabolism, generation of reactive oxygen species (ROS), and Ca(2+) handling to stress responses, cell survival, and death. Malfunction of the organelle may contribute to the pathogenesis of neuromuscular disorders, cancer, premature aging, and cardiovascular diseases, including myocardial ischemia, cardiomyopathy, and heart failure. Mitochondria are unique as they contain their own genome organized into DNA-protein complexes, so-called mitochondrial nucleoids, along with multiprotein machineries, which promote mitochondrial DNA (mtDNA) replication, transcription, and repair. Although the organelle possesses almost all known nuclear DNA repair pathways, including base excision repair, mismatch repair, and recombinational repair, the proximity of mtDNA to the main sites of ROS production and the lack of protective histones may result in increased susceptibility to oxidative stress and other types of mtDNA damage. Defects in the components of these highly organized machineries, which mediate mtDNA maintenance (replication and repair), may result in accumulation of point mutations and/or deletions in mtDNA and decreased mtDNA copy number impairing mitochondrial function. This review will focus on the mechanisms of mtDNA maintenance with emphasis on the proteins implicated in these processes and their functional role in various disease conditions and aging.

Relationship of Mitochondrial-Related Protein Expression with the Differentiation, Metastasis, and Poor Prognosis of Oral Squamous Cell Carcinoma.

Mitochondrial dysfunction and respiratory function changes have been consistently associated with the initiation and progression of cancer. The purpose of this study was to retrospectively investigate the expression of mitochondrial tumor-suppressor and DNA-repair proteins in patients with oral squamous cell carcinoma (OSCC) and to evaluate the relationship between their expression and prognosis. We enrolled 197 patients with OSCC who underwent surgical resection between August 2013 and October 2018. Clinical, pathological, and epidemiological data were retrospectively collected from hospital records. The expression of peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α), mitochondrial transcription factor A, mitochondrial tumor suppressor gene 1, silent information regulator 3, and 8-hydroxyguanine DNA glycosylase was investigated using immunochemistry. The 3-year disease-specific survival (DSS) rates of patients showing positive expression of all selected proteins were significantly higher than those of patients showing a lack of expression. Multivariate analysis revealed that the expression of PGC-1α (hazard ratio, 4.684) and vascular invasion (hazard ratio, 5.690) can predict the DSS rate (p < 0.001). Low PGC-1α expression and vascular invasion are potential clinically effective predictors of the prognosis of OSCC.

In utero ethanol exposure induces mitochondrial DNA damage and inhibits mtDNA repair in developing brain.

Introduction: Mitochondrial dysfunction is postulated to be a central event in fetal alcohol spectrum disorders (FASD). People with the most severe form of FASD, fetal alcohol syndrome (FAS) are estimated to live only 34 years (95% confidence interval, 31 to 37 years), and adults who were born with any form of FASD often develop early aging. Mitochondrial dysfunction and mitochondrial DNA (mtDNA) damage, hallmarks of aging, are postulated central events in FASD. Ethanol (EtOH) can cause mtDNA damage, consequent increased oxidative stress, and changes in the mtDNA repair protein 8-oxoguanine DNA glycosylase-1 (OGG1). Studies of molecular mechanisms are limited by the absence of suitable human models and non-invasive tools. Methods: We compared human and rat EtOH-exposed fetal brain tissues and neuronal cultures, and fetal brain-derived exosomes (FB-Es) from maternal blood. Rat FASD was induced by administering a 6.7% alcohol liquid diet to pregnant dams. Human fetal (11-21 weeks) brain tissue was collected and characterized by maternal self-reported EtOH use. mtDNA was amplified by qPCR. OGG1 and Insulin-like growth factor 1 (IGF-1) mRNAs were assayed by qRT-PCR. Exosomal OGG1 was measured by ddPCR. Results: Maternal EtOH exposure increased mtDNA damage in fetal brain tissue and FB-Es. The damaged mtDNA in FB-Es correlated highly with small eye diameter, an anatomical hallmark of FASD. OGG1-mediated mtDNA repair was inhibited in EtOH-exposed fetal brain tissues. IGF-1 rescued neurons from EtOH-mediated mtDNA damage and OGG1 inhibition. Conclusion: The correlation between mtDNA damage and small eye size suggests that the amount of damaged mtDNA in FB-E may serve as a marker to predict which at risk fetuses will be born with FASD. Moreover, IGF-1 might reduce EtOH-caused mtDNA damage and neuronal apoptosis.

Mitochondrial DNA: Consensuses and Controversies.

In the course of its short history, mitochondrial DNA (mtDNA) has made a long journey from obscurity to the forefront of research on major biological processes. mtDNA alterations have been found in all major disease groups, and their significance remains the subject of intense research. Despite remarkable progress, our understanding of the major aspects of mtDNA biology, such as its replication, damage, repair, transcription, maintenance, etc., is frustratingly limited. The path to better understanding mtDNA and its role in cells, however, remains torturous and not without errors, which sometimes leave a long trail of controversy behind them. This review aims to provide a brief summary of our current knowledge of mtDNA and highlight some of the controversies that require attention from the mitochondrial research community.

POLB: A new role of DNA polymerase beta in mitochondrial base excision repair.

The mitochondrial genome is a matrilineally inherited DNA that encodes numerous essential subunits of the respiratory chain in all metazoans. As such mitochondrial DNA (mtDNA) sequence integrity is vital to organismal survival, but it has a limited cadre of DNA repair activities, primarily base excision repair (BER). We have known that the mtDNA is significantly oxidized by both endogenous and exogenous sources, but this does not lead to the expected preferential formation of transversion mutations, which suggest a robust base excision repair (BER) system. This year, two different groups reported compelling evidence that what was believed to be exclusively nuclear DNA repair polymerase, POLB, is located in the mitochondria and plays a significant role in mitochondrial BER, mtDNA integrity and mitochondrial function. In this commentary, we review the findings and highlight remaining questions for the field.

Mitochondria and mitochondrial DNA as relevant targets for environmental contaminants.

The mitochondrial DNA (mtDNA) is a closed circular molecule that encodes, in humans, 13 polypeptides components of the oxidative phosphorylation complexes. Integrity of the mitochondrial genome is essential for mitochondrial function and cellular homeostasis, and mutations and deletions in the mtDNA lead to oxidative stress, mitochondrial dysfunction and cell death. In vitro and in situ studies suggest that when exposed to certain genotoxins, mtDNA accumulates more damage than nuclear DNA, likely owing to its organization and localization in the mitochondrial matrix, which tends to accumulate lipophilic, positively charged molecules. In that regard, several relevant environmental and occupational contaminants have physical-chemical characteristics that indicate that they might accumulate in mitochondria and target mtDNA. Nonetheless, very little is known so far about mtDNA damage and mitochondrial dysfunction due to environmental exposure, either in model organisms or in humans. In this article, we discuss some of the characteristics of mtDNA which render it a potentially relevant target for damage by environmental contaminants, as well as possible functional consequences of damage/mutation accumulation. In addition, we review the data available in the literature focusing on mitochondrial effects of the most common classes of environmental pollutants. From that, we conclude that several lines of experimental evidence support the idea that mitochondria and mtDNA are susceptible and biologically relevant targets for pollutants, and more studies, including mechanistic ones, are needed to shed more light into the contribution of mitochondrial dysfunction to the environmental and human health effects of chemical exposure.

The "fast" and the "slow" modes of mitochondrial DNA degradation.

In a living cell, oxidative stress resulting from an external or internal insult can result in mitochondrial DNA (mtDNA) damage and degradation. Here, we show that in HeLa cells, mtDNA can withstand relatively high levels of extracellular oxidant H2O2 before it is damaged to a point of degradation, and that mtDNA levels in these cells quickly recover after removal of the stressor. In contrast, mtDNA degradation in mouse fibroblast cells is induced at eight-fold lower concentrations of H2O2, and restoration of the lost mtDNA proceeds much slower. Importantly, mtDNA levels in HeLa cells continue to decline even after withdrawal of the stressor thus marking the "slow" mode of mtDNA degradation. Conversely, in mouse fibroblasts maximal loss of mtDNA is achieved during treatment, and is already detectable at 5 min after exposure, indicating the "fast" mode. These differences may modulate susceptibility to oxidative stress of those organs, which consist of multiple cell types.