Deletion of OGG1 Results in a Differential Signature of Oxidized Purine Base Damage in mtDNA Regions (original) (raw)
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Cancer research, 2001
Mitochondria are not only the major site for generation of reactive oxygen species, but also one of the main targets of oxidative damage. One of the major products of DNA oxidation, 8-oxodeoxyguanosine (8-oxodG), accumulates in mitochondrial DNA (mtDNA) at levels three times higher than in nuclear DNA. The main pathway for the repair of 8-oxodG is the base excision repair pathway initiated by oxoguanine DNA glycosylase (OGG1). We previously demonstrated that mammalian mitochondria from mice efficiently remove 8-oxodG from their genomes and isolated a protein from rat liver mitochondria with 8-oxoguanine (8-oxodG) DNA glycosylase/apurinic DNA lyase activity. In the present study, we demonstrated that the mitochondrial 8-oxodG DNA glycosylase/apurinic DNA lyase activity is the mitochondrial isoform of OGG1. Using mouse liver mitochondria isolated from ogg1(-/-) mice, we showed that the OGG1 gene encodes for the mitochondrial 8-oxodG glycosylase because these extracts have no incision ...
Formation and repair of oxidative damage in the mitochondrial DNA
Mitochondrion, 2014
The mitochondrial DNA (mtDNA) encodes for only 13 polypeptides, components of 4 of the 5 oxidative phosphorylation complexes. But despite this apparently small numeric contribution, all 13 subunits are essential for the proper functioning of the oxidative phosphorylation circuit. Thus, accumulation of lesions, mutations and deletions/insertions in the mtDNA could have severe functional consequences, including mitochondrial diseases, aging and age-related diseases. The DNA is a chemically unstable molecule, which can be easily oxidized, alkylated, deaminated and suffer other types of chemical modifications, throughout evolution the organisms that survived were those who developed efficient DNA repair processes. In the last two decades, it has become clear that mitochondria have DNA repair pathways, which operate, at least for some types of lesions, as efficiently as the nuclear DNA repair pathways. The mtDNA is localized in a particularly oxidizing environment, making it prone to accumulate oxidatively generated DNA modifications (ODMs). In this article, we: i) review the major types of ODMs formed in mtDNA and the known repair pathways that remove them; ii) discuss the possible involvement of other repair pathways, just recently characterized in mitochondria, in the repair of these modifications; and iii) address the role of DNA repair in mitochondrial function and a possible cross-talk with other pathways that may potentially participate in mitochondrial genomic stability, such as mitochondrial dynamics and nuclear-mitochondrial signaling. Oxidative stress and ODMs have been increasingly implicated in disease and aging, and thus we discuss how variations in DNA repair efficiency may contribute to the etiology of such conditions or even modulate their clinical outcomes.
Metabolism and DNA repair shape a specific modification pattern in mitochondrial DNA
Mitochondrion, 2018
The mitochondrial DNA (mtDNA) resides in the vicinity of energy-rich reactions. Thus, chemical modifications of mtDNA might mirror mitochondrial processes and could serve as biomarkers of metabolic processes in the mitochondria. This hypothesis was tested by assessing modifications at 17 different sites in the mtDNA as a function of cell type, oxidative stress and mitochondrial activity. Two mouse mutants with a metabolic phenotype were compared to wild-type (WT) mice: the ogg1-/mouse that lacks the 8-oxoguanine DNA glycosylase (OGG1), and the alkbh7-/mouse missing the ALKBH7 protein that has been implicated in fatty acid oxidation. It was found that cell type, oxidative stress and mitochondrial complex activity shaped distinct modification patterns in mtDNA, and that OGG1 and ALKBH7 independently modulated these modification patterns. The modifications included ribonucleotides, which also accumulated in mtDNA with age. Interestingly, this age-dependent accumulation most likely involves DNA repair, as mtDNA from ogg1-/mice did not accumulate modifications with age. On the other hand, alkbh7-/-mtDNA accumulated more modifications with age than WT mtDNA. Our results show that mtDNA is dynamically modified with metabolic activity and imply a novel synergy between metabolism and mtDNA repair proteins.
Mitochondrial DNA Damage Patterns and Aging: Revising the Evidences for Humans and Mice
Aging and Disease, 2013
A significant body of work, accumulated over the years, strongly suggests that damage in mitochondrial DNA (mtDNA) contributes to aging in humans. Contradictory results, however, are reported in the literature, with some studies failing to provide support to this hypothesis. With the purpose of further understanding the aging process, several models, among which mouse models, have been frequently used. Although important affinities are recognized between humans and mice, differences on what concerns physiological properties, disease pathogenesis as well as life-history exist between the two; the extent to which such differences limit the translation, from mice to humans, of insights on the association between mtDNA damage and aging remains to be established. In this paper we revise the studies that analyze the association between patterns of mtDNA damage and aging, investigating putative alterations in mtDNA copy number as well as accumulation of deletions and of point mutations. Reports from the literature do not allow the establishment of a clear association between mtDNA copy number and age, either in humans or in mice. Further analysis, using a wide spectrum of tissues and a high number of individuals would be necessary to elucidate this pattern. Likewise humans, mice demonstrated a clear pattern of age-dependent and tissuespecific accumulation of mtDNA deletions. Deletions increase with age, and the highest amount of deletions has been observed in brain tissues both in humans and mice. On the other hand, mtDNA point mutations accumulation has been clearly associated with age in humans, but not in mice. Although further studies, using the same methodologies and targeting a larger number of samples would be mandatory to draw definitive conclusions, the revision of the available data raises concerns on the ability of mouse models to mimic the mtDNA damage patterns of humans, a fact with implications not only for the study of the aging process, but also for investigations of other processes in which mtDNA dysfunction is a hallmark, such as neurodegeneration.
Oxidative stress induces degradation of mitochondrial DNA
Nucleic Acids Research, 2009
Mitochondrial DNA (mtDNA) is located in close proximity of the respiratory chains, which are the main cellular source of reactive oxygen species (ROS). ROS can induce oxidative base lesions in mtDNA and are believed to be an important cause of the mtDNA mutations, which accumulate with aging and in diseased states. However, recent studies indicate that cumulative levels of base substitutions in mtDNA can be very low even in old individuals. Considering the reduced complement of DNA repair pathways available in mitochondria and higher susceptibility of mtDNA to oxidative damage than nDNA, it is presently unclear how mitochondria manage to maintain the integrity of their genetic information in the face of the permanent exposure to ROS. Here we show that oxidative stress can lead to the degradation of mtDNA and that strand breaks and abasic sites prevail over mutagenic base lesions in ROS-damaged mtDNA. Furthermore, we found that inhibition of base excision repair enhanced mtDNA degradation in response to both oxidative and alkylating damage. These observations suggest a novel mechanism for the protection of mtDNA against oxidative insults whereby a higher incidence of lesions to the sugar-phosphate backbone induces degradation of damaged mtDNA and prevents the accumulation of mutagenic base lesions.
Oxidative DNA damage stalls the human mitochondrial replisome
Scientific Reports, 2016
Oxidative stress is capable of causing damage to various cellular constituents, including DNA. There is however limited knowledge on how oxidative stress influences mitochondrial DNA and its replication. Here, we have used purified mtDNA replication proteins, i.e. DNA polymerase γ holoenzyme, the mitochondrial single-stranded DNA binding protein mtSSB, the replicative helicase Twinkle and the proposed mitochondrial translesion synthesis polymerase PrimPol to study lesion bypass synthesis on oxidative damage-containing DNA templates. Our studies were carried out at dNTP levels representative of those prevailing either in cycling or in non-dividing cells. At dNTP concentrations that mimic those in cycling cells, the replication machinery showed substantial stalling at sites of damage, and these problems were further exacerbated at the lower dNTP concentrations present in resting cells. PrimPol, the translesion synthesis polymerase identified inside mammalian mitochondria, did not promote mtDNA replication fork bypass of the damage. This argues against a conventional role for PrimPol as a mitochondrial translesion synthesis DNA polymerase for oxidative DNA damage; however, we show that Twinkle, the mtDNA replicative helicase, is able to stimulate PrimPol DNA synthesis in vitro, suggestive of an as yet unidentified role of PrimPol in mtDNA metabolism. The major function of mitochondria is to convert energy from nutrients into ATP through the process of oxidative phosphorylation (OXPHOS). Thirteen genes that encode for essential subunits of the OXPHOS system are located on the mitochondrial DNA (mtDNA), a small circular genome of 16 kb. Therefore it is not surprising that the accumulation of mtDNA molecules with mutations and/or deletions can lead to mitochondrial disease, characterized by decreased energy production 1,2. The process of energy production by the OXPHOS system inevitably generates some reactive oxygen species (ROS) that can cause oxidative damage to cellular components, including DNA. Frequent forms of ROS-induced DNA lesions include abasic sites (AP) and 8-oxo-7,8-dihydroguanine (8-oxo-G) 3. There are several reasons why the effects of ROS on DNA might be especially deleterious in mitochondria. First, it has been proposed that, compared to nuclear DNA, mtDNA has a greater exposure to reactive oxygen species (ROS) due to its association with the mitochondrial inner membrane 4,5 and thus its proximity to the OXPHOS system 6-8. Second, in contrast to nuclear DNA, mtDNA replication is not restricted to S-phase and can therefore also take place when free nucleotide levels are relatively low 9 and the redox environment of the cell is more oxidizing 10 and thus more likely to cause oxidative damage to replicating DNA. These potential problems may be accentuated in quiescent (post-mitotic) cells that do not cycle through S-phase but still duplicate their mtDNA. Previously, the assumed lack of functional DNA repair mechanisms has been considered to contribute to the susceptibility of mtDNA to oxidative damage. However, multiple mtDNA repair pathways, including base excision repair that normally repairs both 8-oxo-G and abasic sites, have been described in the mitochondrial compartment 11,12. MtDNA is replicated by a unique machinery, parts of which are related to their counterparts in phage T7 13,14. The key factors include the heterotrimeric DNA polymerase γ (Pol γ AB 2) that consists of one Pol γ A catalytic subunit and two processivity subunits (Pol γ B), the mitochondrial single-stranded DNA binding protein (mtSSB), the DNA helicase Twinkle and the mitochondrial RNA polymerase (POLRMT) that functions as the primase in mtDNA replication 14. Using these basic components, we have previously been able to reconstitute mtDNA replication in vitro 15,16. During chromosomal DNA replication in the nucleus, both leading and lagging strand replicative polymerases (DNA polymerase ε and δ , respectively) stall when encountering oxidative DNA damage 17 .
Mitochondrial function and mitochondrial DNA maintenance with advancing age
2014
We review the impact of mitochondrial DNA (mtDNA) maintenance and mitochondrial function on the aging process. Mitochondrial function and mtDNA integrity are closely related. In order to create a protective barrier against reactive oxygen and nitrogen species (RONS) attacks and ensure mtDNA integrity, multiple cellular mtDNA copies are packaged together with various proteins in nucleoids. Regulation of antioxidant and RONS balance, DNA base excision repair, and selective degradation of damaged mtDNA copies preserves normal mtDNA quantities. Oxidative damage to mtDNA molecules does not substantially contribute to increased mtDNA mutation frequency; rather, mtDNA replication errors of DNA PolG are the main source of mtDNA mutations. Mitochondrial turnover is the major contributor to maintenance of mtDNA and functionally active mitochondria. Mitochondrial turnover involves mitochondrial biogenesis, mitochondrial dynamics, and selective autophagic removal of dysfunctional mitochondria (i.e., mitophagy). All of these processes exhibit decreased activity during aging and fall under greater nuclear genome control, possibly coincident with the emergence of nuclear genome instability. We suggest that the age-dependent accumulation of mutated mtDNA copies and dysfunctional mitochondria is associated primarily with decreased cellular autophagic and mitophagic activity.