Scientists led by the University of California, Riverside (UC Riverside) have identified a previously unknown form of DNA damage in mitochondria that may underlie a wide range of disorders linked to mitochondrial dysfunction. Their findings have just been published in the Proceedings of the National Academy of Sciences (PNAS).
Mitochondria contain their own DNA (mtDNA), which is essential for the proper functioning of these organelles that convert glucose into ATP, supplying cells with the energy needed to power metabolic processes.
The culprit is a large molecule, glutathionylate, which attaches to DNA and, if left unrepaired, can cause mutations. Researchers at UC Riverside, working with colleagues at the University of Texas MD Anderson Cancer Center, found that glutathionylated mtDNA accumulates in mitochondria at levels up to 80 times higher than in the cell nucleus. In short, the nuclear DNA repair system is vastly more efficient than its mitochondrial counterpart.
For advocates of Intelligent Design (ID), this discovery—if they understood it rather than dismissing it as part of an imagined conspiracy to undermine their faith—creates an acute theological problem. If we temporarily grant the core assumption of ID creationism, that a supernatural designer indistinguishable from the allegedly omniscient, omnipotent, and omnibenevolent god of the Bible and Qur’an is responsible for the design of mitochondrial DNA and its replication machinery, then only two coherent conclusions follow:
- the designer is incompetent, having failed to produce fault-free mtDNA and an adequate repair mechanism, despite supposedly managing this for nuclear DNA; or
- the designer could have produced fault-free mtDNA but chose instead to create error-prone mtDNA and a weak repair process, thereby intentionally designing disease and suffering—in other words, malevolence.
The notion of an omniscient designer also rules out the excuse that the harmful consequences were unforeseeable. An all-knowing creator would have foreseen them; yet, according to ID logic, the designer implemented them regardless—designing mitochondrial DNA to fail and cause disease.
Thus, a biological phenomenon that fits seamlessly within the framework of evolutionary theory becomes an insurmountable theological obstacle for ID advocates, who must contort the evidence to suit a predetermined conclusion while catering to a scientifically illiterate and credulous audience.
The Evolutionary Origin of Mitochondria. Mitochondria are widely understood to have originated through a landmark event in early cellular evolution known as *endosymbiosis*. Around 1.5–2 billion years ago, an ancestral archaeal cell is thought to have engulfed a free-living bacterium—most likely an alphaproteobacterium—without digesting it. Instead, the two organisms formed a stable, mutually beneficial relationship.The UC Riverside team’s research is summarised in a UC Riverside news item by Iqbal Pittalwala.
The host cell provided protection and access to nutrients, while the bacterium contributed a highly efficient system for aerobic respiration, producing large amounts of ATP. Over time, this partnership became permanent. The bacterium transferred most of its genes to the host nucleus, losing the ability to live independently. The few genes that remained within the bacterium’s own genome evolved into what we now recognise as mitochondrial DNA (mtDNA).
This process created the first eukaryotic cells—cells with internal compartments—and set the stage for the evolution of all complex life, from algae and fungi to plants and animals.
Modern mitochondria retain several hallmarks of their bacterial ancestry:
- They have their own circular DNA, resembling bacterial genomes.
- They divide by binary fission, like bacteria.
- Their membranes and ribosomes show clear bacterial affinities.
Thus, mitochondria are best understood not as purpose-built “energy factories”, but as domesticated symbionts whose integration into early eukaryotes allowed life to expand into new ecological niches and evolve unprecedented complexity.
New type of DNA damage found in our cells’ powerhouses
Findings may have important implications for diseases associated with mitochondrial problems
A previously unknown type of DNA damage in the mitochondria, the tiny power plants inside our cells, could shed light on how our bodies sense and respond to stress. The findings of the UC Riverside-led study are published today in the Proceedings of the National Academy of Sciences and have potential implications for a range of mitochondrial dysfunction-associated diseases, including cancer and diabetes.
Mitochondria have their own genetic material, known as mitochondrial DNA (mtDNA), which is essential for producing the energy that powers our bodies and sending signals within and outside cells. While it has long been known that mtDNA is prone to damage, scientists didn't fully understand the biological processes. The new research identifies a culprit: glutathionylated DNA (GSH-DNA) adducts.
An adduct is bulky chemical tag formed when a chemical, such as a carcinogen, attaches directly to DNA. If the damage isn’t repaired, it can lead to DNA mutations and increase the risk of disease.
A “sticky” problem for mitochondrial DNA
The researchers found in their experiments in cultured human cells that these adducts accumulate at levels up to 80 times higher in mtDNA than in the DNA of the cell’s nucleus, suggesting that mtDNA is particularly vulnerable to this type of damage.
Linlin Zhao, senior author and an associate professor of chemistry at UCR, explained that mtDNA makes up only a small fraction — about 1-5% — of all the DNA in a cell. It is circular in shape, has just 37 genes, and is passed down only from the mother. In contrast, nuclear DNA (nDNA) is linear in shape and inherited from both parents.
mtDNA is more prone to damage than nDNA. Each mitochondrion has many copies of mtDNA, which provides some backup protection. The repair systems for mtDNA are not as strong or efficient as those for nuclear DNA.
Associate Professor Linlin Zhao, senior author
Department of Chemistry
University of California, Riverside, CA, USA.
Lead researcher and first author, Yu Hsuan Chen, a doctoral student in Zhao’s lab, likened the mitochondrion to the cell’s engine and signaling hub.When the engine’s manual — the mtDNA — gets damaged, it’s not always by a spelling mistake, a mutation. Sometimes, it’s more like a sticky note that gets stuck to the pages, making it hard to read and use. That’s what these GSH-DNA adducts are doing.
Yu Hsuan Chen, first author
Department of Chemistry
University of California, Riverside, CA, USA.
From DNA damage to disease
The researchers linked the accumulation of the sticky lesions to significant changes in mitochondrial function. They observed a decrease in proteins needed for energy production and a simultaneous increase in proteins that help with stress response and mitochondrial repair, suggesting the cell fights back against the damage.
The researchers also used advanced computer simulations to model the effect of the adducts.
We found that the sticky tags can actually make the mtDNA less flexible and more rigid. This might be a way the cell ‘marks’ damaged DNA for disposal, preventing it from being copied and passed on.
Yu Hsuan Chen.
The team’s findings hold promise for understanding diseases. According to Zhao, the discovery of GSH-DNA adducts opens a new frontier for research into how damaged mtDNA can act as a stress signal.
Problems with mitochondria and inflammation linked to damaged mtDNA have been connected to diseases such as neurodegeneration and diabetes. When mtDNA is damaged, it can escape from the mitochondria and trigger immune and inflammatory responses. The new type of mtDNA modification we’ve discovered could open new research directions to understand how it influences immune activity and inflammation.
Associate Professor Linlin Zhao.
Publication:
The discovery that mitochondrial DNA accumulates glutathionylation damage at levels dozens of times higher than nuclear DNA further exposes the inherent fragility of mitochondria. If one assumes an omniscient, omnipotent, and omnibenevolent designer, this arrangement is extraordinarily difficult to justify. A competent designer would not create two essential DNA systems—nuclear and mitochondrial—with such starkly different capacities for repair, nor would it embed a critical energy-producing system with a known propensity for failure and disease. The notion that an all-knowing creator would deliberately engineer a repair mechanism that leaves mitochondrial DNA so vulnerable raises obvious questions about either the competence or the intentions of such a being.Significance
Mitochondrial DNA (mtDNA) plays a crucial role in energy metabolism, cellular signaling, and human disease development. While it is known that mtDNA is susceptible to damage, the biological consequences of this damage remain partially understood. We report a type of mtDNA damage, glutathionylated DNA (GSH-DNA) adducts. These adducts form from endogenous abasic sites and alkylation DNA damage, catalyzed by a mtDNA packing protein (TFAM) and polyamines. Our study clarifies their accumulation, regulation, and biological effects in mitochondria. Given the emerging role of mtDNA and a commonly used oxidative DNA damage marker, 8-oxo-7,8-dihydro-2’-deoxyguanosine, in promoting inflammation, the high levels of GSH-DNA adducts in mtDNA highlight the critical need to investigate their roles in innate immune and inflammatory diseases.
Abstract
Mitochondrial DNA (mtDNA) is crucial for cellular energy production, metabolism, and signaling. Its dysfunction is implicated in various diseases, including mitochondrial disorders, neurodegeneration, and diabetes. mtDNA is susceptible to damage by endogenous and environmental factors; however, unlike nuclear DNA (nDNA), mtDNA lesions do not necessarily lead to an increased mutation load in mtDNA. Instead, mtDNA lesions have been implicated in innate immunity and inflammation. Here, we report a type of mtDNA damage: glutathionylated DNA (GSH-DNA) adducts. These adducts are formed from abasic (AP) sites, key intermediates in base excision repair, or from alkylation DNA damage. Using mass spectrometry, we quantified the GSH-DNA lesion in both nDNA and mtDNA and found its significant accumulation in mtDNA of two different human cell lines, with levels one or two orders of magnitude higher than in nDNA. The formation of GSH-DNA adducts is influenced by TFAM and polyamines, and their levels are regulated by repair enzymes AP endonuclease 1 (APE1) and tyrosyl-DNA phosphodiesterase 1 (TDP1). The accumulation of GSH-DNA adducts is associated with the downregulation of several ribosomal and complex I subunit proteins and the upregulation of proteins related to redox balance and mitochondrial dynamics. Molecular dynamics (MD) simulations revealed that the GSH-DNA lesion stabilizes the TFAM-DNA binding, suggesting shielding effects from mtDNA transactions. Collectively, this study provides critical insights into the formation, regulation, and biological effects of GSH-DNA adducts in mtDNA. Our findings underscore the importance of understanding how these lesions may contribute to innate immunity and inflammation.
In higher eukaryotes, mitochondria are indispensable organelles for cellular metabolism and signaling. Mitochondria house a multi-copy and circular genome, known as mitochondrial DNA (mtDNA). The relatively small (16,569 bp) genome encodes essential protein subunits of the oxidative phosphorylation system, as well as a suite of tRNAs and rRNAs, critical for mitochondrial function (1, 2). Beyond its gene-encoding role, mtDNA can also act as a damage-associated molecular pattern (DAMP) molecule upon release into the cytoplasm, triggering immune and inflammatory responses (3, 4). Consequently, mtDNA defects are implicated in a spectrum of pathologies, including mitochondrial disorders, neurodegenerative diseases, and aging (5, 6).
mtDNA exists in multiple copies and exhibits heteroplasmy, a state of coexistence of normal and defective mtDNA molecules harboring mutations and deletions. While mitochondria can maintain regular function below a certain threshold of mtDNA heteroplasmy, mtDNA is inherently susceptible to oxidative damage and environmental insults (7–9), leading to the formation of covalent DNA adducts or lesions. Due to its redundancy and limited repair mechanisms, mtDNA accumulates DNA lesions at higher levels than nuclear DNA (nDNA) upon exposure to genotoxic compounds (9). Intriguingly, the increased lesion burden often does not translate to elevated mutation rates (10, 11), but rather a rapid mtDNA copy number decline (9), suggesting active degradation and release of damaged mtDNA molecules (2, 12). Indeed, protein factors, such as DNA polymerase γ (13, 14), mitochondrial genome maintenance exonuclease 1 (MGME1) (13, 15, 16), and mitochondrial transcription factor A (TFAM) (17, 18), have been shown to be involved in mtDNA degradation with different types of mtDNA damage. Furthermore, both native and damaged mtDNA can be extruded from mitochondria, triggering innate immune and inflammatory responses via pattern recognition receptors (3, 4) in the cytoplasm. mtDNA containing certain DNA lesions, such as 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), exhibits enhanced proinflammatory potential (19–21), suggesting a role of mtDNA lesions beyond mutagenesis. The heightened vulnerability of mtDNA and its immunogenic potential have led to its proposed function as a genotoxic stress sensor (22, 23).
In human mitochondria, base excision repair (BER) is the primary DNA repair pathway for removing nucleobase lesions and single-strand breaks (SSBs) (24, 25). This process, analogous to nuclear BER, involves damaged base excision by DNA glycosylases, AP site cleavage by apurinic/apyrimidinic endonuclease 1 (APE1), 5'-deoxyribose phosphate (5'-dRp) removal, nucleotide insertion, and ligation. A critical BER intermediate is the abasic (AP) site, a highly reactive lesion that, if unrepaired, can lead to strand breaks and mutations (26, 27). AP sites can undergo β-elimination, generating 3'-phospho-α,β-unsaturated aldehyde (3'-PUA) residues, which can react with cellular thiols, particularly glutathione (GSH), forming glutathionylated DNA (GSH-DNA) adducts (28–30). While GSH-DNA adducts have been detected in nDNA (30), their biological effects in the nucleus remain unknown. The formation of these relatively bulky adducts raises questions regarding their structural and biological impacts, especially considering the known protective roles of GSH in scavenging other toxic electrophiles, such as oxidative DNA damage products, base propenals (31), and lipid peroxidation byproducts (acrolein and 4-hydroxy-2-nonenal) (32).
Given the prevalence of mtDNA lesions and their potential signaling roles, quantifying GSH-DNA adducts in mtDNA and elucidating their biological implications are crucial. In this study, we clarified the level, regulation, and biological effects of GSH-DNA adducts in mtDNA. Using isotope dilution mass spectrometry, we quantified GSH-DNA adduct levels in both nDNA and mtDNA from cultured human cells under various genotoxic stress conditions. Our results demonstrate a preferential accumulation of GSH-DNA adducts in mtDNA compared to nDNA in two different cell lines under various conditions, with 13- to 300-fold higher levels in mtDNA than nDNA. We examined the roles of APE1 and tyrosyl-DNA phosphodiesterase 1 (TDP1), two key enzymes in 3'DNA lesion repair, revealing their respective roles in preventing GSH-DNA adduct accumulation. In addition, the GSH-DNA adduct formation is catalyzed by TFAM and polyamines and regulated by local TFAM concentrations. The accumulation of GSH-DNA adducts is associated with downregulation of several ribosomal and complex I subunit proteins and upregulation of proteins related to redox balance and mitochondrial dynamics. Furthermore, GSH-DNA adducts enhance conformational flexibility of DNA and stabilize TFAM-DNA complexes, as revealed in molecular dynamics (MD) simulations. Together, our findings provide critical insights into the accumulation, regulation, and biological implications of GSH-DNA adducts in mtDNA. Considering that the abundance of GSH-DNA adducts in mtDNA is comparable to the commonly used oxidative DNA damage marker, 8-oxodG, and the emerging role of mitochondrial 8-oxodG in inflammatory response (19–21), our findings implicate a role of mitochondrial GSH-DNA adducts in innate immunity and inflammation.
Fig. 1.
Formation and quantification of GSH-DNA adducts in HeLa cells. (A) Mechanism of formation of GSH-DNA adducts and TFAM DNA–protein cross-links (TFAM-DPCs) from abasic (AP) sites. DPCin indicates a DPC structure with an uncleaved DNA strand. DPCcl indicates a DPC structure with a cleaved DNA strand upon β-elimination. (B) Representative selected-ion chromatograms (SICs) of GS-ddR-MX and [13C2, 15N]-GS-ddR-MX from mass spectrometry analysis. MX, methoxyamine. (C) Quantification of GSH-DNA adducts, determined by integrated peak area ratios in mass spectrometry analysis, in HeLa cells without or with induced (by doxycycline for 24 h) mitochondrial AP sites. Data represent mean ± SD (n = 3 to 5). Statistical significance was calculated using a one-way ANOVA followed by a Tukey’s HSD (Honestly Significant Difference) post hoc test. *P < 0.05, **P < 0.01, ***P < 0.001. ****P < 0.0001.
Y.H. Chen, M. Esparza Sanchez, T.I. Hung, J. Tang, W. Xu, J. Yin, Y. Wang, C.A. Chang, H. Zhang, J. Chen, & L. Zhao
Glutathionylated DNA adducts accumulate in mitochondrial DNA and are regulated by AP endonuclease 1 and tyrosyl-DNA phosphodiesterase 1 Proc. Natl. Acad. Sci. U.S.A. 122 (47) e2509312122, https://doi.org/10.1073/pnas.2509312122 (2025).
Copyright: © 2025 The authors.
Published by the National Academy of Sciences of the USA. Open access.
Reprinted under a Creative Commons Attribution 4.0 International license (CC BY 4.0)
By contrast, this discovery fits seamlessly into the scientific account of how mitochondria arose. As descendants of free-living bacteria brought into early eukaryotic cells through endosymbiosis, mitochondria retained only a small remnant of their original genome. Over billions of years, most of their genes were transferred to the host nucleus, and the mitochondrial environment—rich in reactive oxygen species and lacking the sophisticated repair pathways that evolved in the nucleus—became a natural hotspot for damage. Evolution does not work toward perfection; it works with what is available, producing workable compromises rather than flawless engineering. The vulnerability of mtDNA is exactly what one would expect from a system cobbled together through historical contingency rather than intention.
The sharp disparity in DNA repair efficiency between the nucleus and mitochondria is therefore not a theological puzzle but an evolutionary inevitability. Evolutionary processes readily explain why the mitochondrial genome survives despite being sub-optimal: it persists because it is just good enough, and because the cost of transferring its remaining genes to the nucleus or completely redesigning its replication machinery exceeds what selection pressures demand. In this light, the newly identified glutathionylation damage is simply another example of evolutionary baggage—features inherited, constrained, and imperfect, reflecting the blind, incremental nature of natural selection rather than the deliberate work of an all-powerful designer.
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