Vanderbilt Institute of Chemical Biology



Discovery at the VICB







A Closer Look at Mitochondrial DNA Damage


By: Carol A. Rouzer, VICB Communications
Published:  February 22, 2018



High levels of the M1dG adduct in mitochondrial DNA are correlated with organelle-associated oxidative stress.     


Reactive oxygen species (ROS) that are produced during physiological and pathological processes play a role in both homeostatic signaling and the inflammatory response. Due to their ability to react with proteins, lipids, and nucleic acids, ROS in high concentrations can also lead to toxicity. Among the products of the reaction of ROS with lipids and DNA are malondialdehyde (MDA) and base propenals, respectively. These electrophilic α,β-unsaturated aldehydes can in turn react with DNA, producing primarily the exocyclic adduct M1dG (Figure 1). The presence of M1dG in the DNA isolated from cells and intact animals, the correlation of its levels with oxidative stress, and evidence of its mutagenicity and carcinogenicity, attest to the adduct's importance to health and disease. Now, Vanderbilt Institute of Chemical Biology member Larry Marnett and his laboratory report the presence of unexpectedly high levels of M1dG in mitochondrial DNA (mtDNA) [O. R. Wauchope, et al. Nucl. Acids Res., (2018) published online Feb. 9, DOI:10.1093/nar/gky089].


M1dG levels in the mtDNA isolated from multiple different cell lines were consistently two orders of magnitude higher than those in the nuclear DNA of the same cells. Particularly notable was the finding that M1dG levels in both nuclear DNA and mtDNA were about 10-fold higher in RAW264.7 cells than in the other cell lines tested. RAW264.7 is a macrophage-like cell line that is frequently used as a model of inflammatory signaling.

Prior work had shown that M1dG in nuclear DNA is removed by nucleotide excision repair (NER), leading to the release of the free M1dG nucleoside. The nucleoside is then subject to oxidation by xanthine oxidase or aldehyde oxidase to form 6-oxo-M1dG (Figure 1), which is excreted in the feces and urine. More recently, the Marnett lab investigators showed that M1dG in intact DNA can also be oxidized to 6-oxo-M1dG by a process that appears to be enzymatic. Indeed, treatment of cells with adenine propenal resulted in a substantial increase in M1dG levels that then gradually declined and were replaced by a stoichiometric amount of 6-oxo-M1dG. In contrast, although M1dG levels in mtDNA also increased following adenine propenal treatment, they remained constant thereafter. No 6-oxo-M1dG was detected in mtDNA, either basally or following exposure to adenine propenal.



FIGURE 1. Formation of M1dG and 6-oxo-M1dG. Malondialdehyde (MDA) (formed by lipid peroxidation) or base propenals (formed by direct oxidation of DNA) react with dG bases in DNA to form M1dG. Repair of M1dG by nucleotide excision repair (NER) leads to release of the free M1dG nucleoside, which can then be oxidized by aldehyde oxidase or xanthine oxidase to yield 6-oxo-M1dG. Alternatively, M1dG in nuclear DNA can be oxidized by an unknown enzyme to form 6-oxo-M1dG in DNA.



The high levels of M1dG in mtDNA led the investigators to hypothesize that ROS generated during mitochondrial respiration may contribute to formation of the adduct. To test this hypothesis, they exposed cells to rotenone, a respiratory complex I inhibitor that promotes formation of superoxide in mitochondria. Consistent with their hypothesis, rotenone-treated cells exhibited substantially higher levels of M1dG in mtDNA but not nuclear DNA. Also consistent with the hypothesis, the scavenger of mitochondrial superoxide mitoTEMPO blocked the rotenone-mediated increase in M1dG formation, whereas the generalized superoxide scavenger TEMPO did not.


The researchers next tested the hypothesis that some sequences in mtDNA are more susceptible to M1dG formation than others. They isolated mtDNA from cells that had or had not been exposed to adenine propenal, fragmented the DNA, and used affinity chromatography with an antibody directed against M1dG to selectively capture fragments containing the adduct (Figure 2). Determination of the sequences of the captured fragments revealed that M1dG was uniformly distributed across the mitochondrial genome regardless of prior exposure to adenine propenal. Thus, there appears to be no sequence specificity for M1dG formation in mtDNA.



FIGURE 2. Protocol for isolating mtDNA fragments containing M1dG. Following isolation, the mtDNA is fragmented and then incubated with beads coated with an antibody directed towards M1dG. Fragments bound to the antibody are eluted and subjected to sequence analysis. Figure reproduced under the Creative Commons Attribution-NonCommercial 4.0 International CC BY-NC License from O. R. Wauchope, et al. Nucl. Acids Res., (2018) published online Feb. 9, DOI:10.1093/nar/gky089.


Bone morphogenic proteins (BMPs) comprise a group of growth factors originally noted for their ability to promote bone and cartilage formation. We know now that BMPs play a role in many aspects of cell differentiation and development. In fact, people who carry a rare mutation in the gene encoding a BMP receptor (BMPR2) suffer from pulmonary arterial hypertension, a lung disease characterized by high blood pressure in the lungs, shortness of breath, poor exercise tolerance, dizziness, and chest pain. Aberrant function of BMPR2 is also associated with increased ROS production and oxidative stress in the mitochondria, leading the investigators to hypothesize that increased M1dG levels might be present in mtDNA from the lungs of people carrying the BMPR2 mutation. As human samples were not available to test this hypothesis, the investigators turned to a transgenic mouse model that carries a comparable BMPR2 mutation and exhibits pulmonary arterial hypertension. They found that mtDNA M1dG levels were higher in pulmonary microvascular endothelial cells from mice bearing the mutation than in those from wild-type mice. This is the first clear association of a specific form of mtDNA damage with a disease state.

The results confirm the correlation between M1dG formation and oxidative stress and demonstrate a high susceptibility of mtDNA to this form of damage. Undoubtedly, the high levels of M1dG in mtDNA are partially attributable to the formation of ROS associated with mitochondrial respiration. However, the absence of the NER pathway and the failure to convert M1dG to 6-oxo-M1dG in mtDNA likely also contribute to the high steady state levels of the adduct in mtDNA. Further work will be required to completely understand the role, if any, that mtDNA-associated M1dG plays in health and disease.




View Necleic Acids Res. article: Oxidative stress increases M1dG, a major peroxidation-derived DNA adduct, in mitochondrial DNA







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