Mitochondrial Base Excision Repair Research Articles

The Role of Poly(ADP-ribose) Polymerase 1 in Nuclear and Mitochondrial Base Excision Repair.

Poly(ADP-ribose) (PAR) Polymerase 1 (PARP-1), also known as ADP-ribosyl transferase with diphtheria toxin homology 1 (ARTD-1), is a critical player in DNA damage repair, during which it catalyzes the ADP ribosylation of self and target enzymes. While the nuclear localization of PARP-1 has been well established, recent studies also suggest its mitochondrial localization. In this review, we summarize the differences between mitochondrial and nuclear Base Excision Repair (BER) pathways, the involvement of PARP-1 in mitochondrial and nuclear BER, and its functional interplay with other BER enzymes.

Targeting mitochondrial DNA polymerase gamma for selective inhibition of MLH1 deficient colon cancer growth.

Synthetic lethality in DNA repair pathways is an important strategy for the selective treatment of cancer cells without harming healthy cells and developing cancer-specific drugs. The synthetic lethal interaction between the mismatch repair (MMR) protein, MutL homolog 1 (MLH1), and the mitochondrial base excision repair protein, DNA polymerase γ (Pol γ) was used in this study for the selective treatment of MLH1 deficient cancers. Germline mutations in the MLH1 gene and aberrant MLH1 promoter methylation result in an increased risk of developing many cancers, including nonpolyposis colorectal and endometrial cancers. Because the inhibition of Pol γ in MLH1 deficient cancer cells provides the synthetic lethal selectivity, we conducted a comprehensive small molecule screening from various databases and chemical drug library molecules for novel Pol γ inhibitors that selectively kill MLH1 deficient cancer cells. We characterized these Pol γ inhibitor molecules in vitro and in vivo, and identified 3,3’-[(1,1’-Biphenyl)-4’,4’-diyl)bis(azo)]bis[4-amino-1-naphthalenesulfonic acid] (congo red; CR; Zinc 03830554) as a high-affinity binder to the Pol γ protein and potent inhibitor of the Pol γ strand displacement and one-nucleotide incorporation DNA synthesis activities in vitro and in vivo. CR reduced the cell proliferation of MLH1 deficient HCT116 human colon cancer cells and suppressed HCT116 xenograft tumor growth whereas it did not affect the MLH1 proficient cell proliferation and xenograft tumor growth. CR caused mitochondrial dysfunction and cell death by inhibiting Pol γ activity and oxidative mtDNA damage repair, increasing the production of reactive oxygen species and oxidative mtDNA damage in MLH1 deficient cells. This study suggests that the Pol γ inhibitor, CR may be further evaluated for the MLH1 deficient cancers’ therapy.

Sirt3 Regulates Response to Oxidative Stress by Interacting with BER Proteins in Colorectal Cancer.

The oxidative damages are well-recognized factors in the pathogenesis of colorectal cancer (CRC). Increased levels of reactive oxygen species (ROS) can lead to oxidative DNA damage, which, if unrepaired, can be an underlying cause of cancerogenic transformation. To defend against these threats, cells have developed a range of defense mechanisms. One of the most important protection mechanisms is DNA repair systems, both nuclear and mitochondrial. Sirt3 is a mitochondrial protein involved in regulating NEIL1, NEIL2, MUTYH, APE1, and LIG3 proteins, which are involved in DNA repair, including mitochondrial repair through mtBER (mitochondrial Base Excision Repair). In this work, we show that NEIL1, NEIL2, MUTYH, APE1, and LIG3 are regulated by Sirt3 through deacetylation, and moreover, Sirt3 is directly involved in physical interaction with MUTYH, NEIL1, and APE1, which indicates the controlling role of Sirt3 over the mtBER mechanism. Also, if the cells deprived of Sirt3 are exposed to oxidative stress, altered levels of those proteins can be observed, which supports the theory of the regulatory role of Sirt3. Finally, to fully confirm the role of Sirt3 in DNA repair, we examined its role in apoptosis and found the impact of this protein on cell survival rate. Using the knowledge obtained in the course of conducted experiments, we postulate consideration of Sirt3 as a target in the rising vulnerability of cancer cells during therapy and therefore increasing the effectiveness of cancer treatment.

Fluorescence Imaging of Mitochondrial DNA Base Excision Repair Reveals Dynamics of Oxidative Stress Responses.

Mitochondrial function in cells declines with aging and with neurodegeneration, due in large part to accumulated mutations in mitochondrial DNA (mtDNA) that arise from deficient DNA repair. However, measuring this repair activity is challenging. We employ a molecular approach for visualizing mitochondrial base excision repair (BER) activity in situ by use of a fluorescent probe (UBER) that reacts rapidly with AP sites resulting from BER activity. Administering the probe to cultured cells revealed signals that were localized to mitochondria, enabling selective observation of mtDNA BER intermediates. The probe showed elevated DNA repair activity under oxidative stress, and responded to suppression of glycosylase activity. Furthermore, the probe illuminated the time lag between the initiation of oxidative stress and the initial step of BER. Absence of MTH1 in cells resulted in elevated demand for BER activity upon extended oxidative stress, while the absence of OGG1 activity limited glycosylation capacity.

Fluorescence Imaging of Mitochondrial DNA Base Excision Repair Reveals Dynamics of Oxidative Stress Responses

AbstractMitochondrial function in cells declines with aging and with neurodegeneration, due in large part to accumulated mutations in mitochondrial DNA (mtDNA) that arise from deficient DNA repair. However, measuring this repair activity is challenging. We employ a molecular approach for visualizing mitochondrial base excision repair (BER) activity in situ by use of a fluorescent probe (UBER) that reacts rapidly with AP sites resulting from BER activity. Administering the probe to cultured cells revealed signals that were localized to mitochondria, enabling selective observation of mtDNA BER intermediates. The probe showed elevated DNA repair activity under oxidative stress, and responded to suppression of glycosylase activity. Furthermore, the probe illuminated the time lag between the initiation of oxidative stress and the initial step of BER. Absence of MTH1 in cells resulted in elevated demand for BER activity upon extended oxidative stress, while the absence of OGG1 activity limited glycosylation capacity.

The Influence of 5',8-Cyclo-2'-deoxypurines on the Mitochondrial Repair of Clustered DNA Damage in Xrs5 Cells: The Preliminary Study.

The 5′,8-cyclo-2′-deoxypurines (cdPus) affect the DNA structure. When these bulky structures are a part of clustered DNA lesions (CDL), they affect the repair of the other lesions within the cluster. Mitochondria are crucial for cell survival and have their own genome, hence, are highly interesting in the context of CDL repair. However, no studies are exploring this topic. Here, the initial stages of mitochondrial base excision repair (mtBER) were considered—the strand incision and elongation. The repair of a single lesion (apurinic site (AP site)) accompanying the cdPu within the double-stranded CDL has been investigated for the first time. The type of cdPu, its diastereomeric form, and the interlesion distance were taken into consideration. For these studies, the established experimental model of short oligonucleotides (containing AP sites located ≤7 base pairs to the cdPu in both directions) and mitochondrial extracts of the xrs5 cells were used. The obtained results have shown that the presence of cdPus influenced the processing of an AP site within the CDL. Levels of strand incision and elongation were higher for oligos containing RcdA and ScdG than for those with ScdA and RcdG. Investigated stages of mtBER were more efficient for DNA containing AP sites located on 5′-end side of cdPu than on its 3′-end side. In conclusion, the presence of cdPus in mtDNA structure may affect mtBER (processing the second mutagenic lesion within the CDL). As impaired repair processes may lead to serious biological consequences, further studies concerning the mitochondrial repair of CDL are highly demanded.

Plasmodium Ape1 is a multifunctional enzyme in mitochondrial base excision repair and is required for efficient transition from liver to blood stage infection

The malaria parasite has a single mitochondrion which carries multiple tandem repeats of its 6 kb genome encoding three proteins of the electron transport chain. There is little information about DNA repair mechanisms for mitochondrial genome maintenance in Plasmodium spp. Of the two AP-endonucleases of the BER pathway encoded in the parasite nuclear genome, the EndoIV homolog PfApn1 has been identified as a mitochondrial protein with restricted functions. We explored the targeting and biochemical properties of the ExoIII homolog PfApe1. PfApe1 localized in the mitochondrion and exhibited AP-site cleavage, 3′-5′ exonuclease, 3′-phosphatase, nucleotide incision repair (NIR) and RNA cleavage activities indicating a wider functional role than PfApn1. The parasite enzyme differed from human APE1 in possessing a large, disordered N-terminal extension. Molecular modelling revealed conservation of structural domains but variations in DNA-interacting residues and an insertion in the α-8 loop suggested differences with APE1. Unlike APE1, where AP-site cleavage and NIR activities could be mutually exclusive based on pH and Mg2+ ion concentration, PfApe1 was optimally active under similar conditions suggesting that it can function both as an AP-endonuclease in BER and directly cleave damaged bases in NIR under similar physiological conditions. To investigate the role of Ape1 in malaria life cycle, we disrupted the gene by double-cross-over homologous recombination. Ape1 knockout (KO) P. berghei parasites showed normal development of blood and mosquito stages. However, inoculation of mice with Ape1 KO salivary gland sporozoites revealed a reduced capacity to initiate blood stage infection. Ape1 KO parasites underwent normal liver stage development until merozoites egressed from hepatocytes. Our results indicated that the delay in pre-patent period was due to the inability of Ape1 KO merosomes to infect erythrocytes efficiently.

DNA polymerase β outperforms DNA polymerase γ in key mitochondrial base excision repair activities

DNA polymerase beta (POLβ), well known for its role in nuclear DNA base excision repair (BER), has been shown to be present in the mitochondria of several different cell types. Here we present a side-by-side comparison of BER activities of POLβ and POLγ, the mitochondrial replicative polymerase, previously thought to be the only mitochondrial polymerase. We find that POLβ is significantly more proficient at single-nucleotide gap filling, both in substrates with ends that require polymerase processing, and those that do not. We also show that POLβ has a helicase-independent functional interaction with the mitochondrial helicase, TWINKLE. This interaction stimulates strand-displacement synthesis, but not single-nucleotide gap filling. Importantly, we find that purified mitochondrial extracts from cells lacking POLβ are severely deficient in processing BER intermediates, suggesting that mitochondrially localized DNA POLβ may be critical for cells with high energetic demands that produce greater levels of oxidative stress and therefore depend upon efficient BER for mitochondrial health.

Hippocampal tau oligomerization early in tau pathology coincides with a transient alteration of mitochondrial homeostasis and DNA repair in a mouse model of tauopathy

Insoluble intracellular aggregation of tau proteins into filaments and neurodegeneration are histopathological hallmarks of Alzheimer disease (AD) and other tauopathies. Recently, prefibrillar, soluble, oligomeric tau intermediates have emerged as relevant pathological tau species; however, the molecular mechanisms of neuronal responses to tau oligomers are not fully understood. Here, we show that hippocampal neurons in six-month-old transgenic mouse model of tauopathy, THY-Tau22, are enriched with oligomeric tau, contain elongated mitochondria, and display cellular stress, but no overt cytotoxicity compared to the control mice. The levels of several key mitochondrial proteins were markedly different between the THY-Tau22 and control mice hippocampi including the mitochondrial SIRT3, PINK1, ANT1 and the fission protein DRP1. DNA base excision repair (BER) is the primary defense system against oxidative DNA damage and it was elevated in six-month-old transgenic mice. DNA polymerase β, the key BER DNA polymerase, was enriched in the cytoplasm of hippocampal neurons in six-month-old transgenic mice and localized with and within mitochondria. Polβ also co-localized with mitochondria in human AD brains in neurons containing oligomeric tau. Most of these altered mitochondrial and DNA repair events were specific to the transgenic mice at 6 months of age and were not different from control mice at 12 months of age when tau pathology reaches its maximum and oligomeric forms of tau are no longer detectable. In summary, our data suggests that we have identified key cellular stress responses at early stages of tau pathology to preserve neuronal integrity and to promote survival. To our knowledge, this work provides the first description of multiple stress responses involving mitochondrial homeostasis and BER early during the progression of tau pathology, and represents an important advance in the etiopathogenesis of tauopathies.

Mitochondrial base excision repair positively correlates with longevity in the liver and heart of mammals.

Damage to DNA is especially important for aging. High DNA repair could contribute, in principle, to lower such damage in long-lived species. However, previous studies showed that repair of endogenous damage to nuclear DNA (base excision repair, BER) is negatively or not correlated with mammalian longevity. However, we hypothesize here that mitochondrial, instead of nuclear, BER is higher in long-lived than in short-lived mammals. We have thus measured activities and/or protein levels of various BER enzymes including DNA glycosylases, NTHL1 and NEIL2, and the APE endonuclease both in total and mitochondrial liver and heart fractions from up to eight mammalian species differing by 13-fold in longevity. Our results show, for the first time, a positive correlation between (mitochondrial) BER and mammalian longevity. This suggests that the low steady-state oxidative damage in mitochondrial DNA of long-lived species would be due to both their lower mitochondrial ROS generation and their higher mitochondrial BER. Long-lived mammals do not need to continuously maintain high nuclear BER levels because they release less mitROS to the cytosol. This can be the reason why they tend to show lower nuclear BER values. The higher mitochondrial BER of long-lived mammals contributes to their superior longevity, agrees with the updated version of the mitochondrial free radical theory of aging, and indicates the special relevance of mitochondria and mitROS for aging.

Plasmodiumfalciparum Apn1 homolog is a mitochondrial base excision repair protein with restricted enzymatic functions.

The malaria parasite carries two organelles, the apicoplast and mitochondrion, whose DNA genomes must be maintained for optimal function and parasite survival under genotoxic stress. DNA repair mechanism(s) operative within these organelles were explored by mining the Plasmodiumfalciparum nuclear genome for sequences encoding proteins of major DNA repair pathways with predicted targeting to either organelle. Of the panel of enzymes identified for base excision repair (BER), we characterized the apurinic/apyrimidinic (AP) endonuclease PfApn1-an EndoIV whose homolog is not known in humans. PfApn1 targeted to the mitochondrion and functioned as an AP endonuclease requiring both Zn2+ and Mn2+ ions for maximal activity. Mutation of the critical third metal-binding site residue H542 resulted in the loss of Mn2+ (but not Zn2+ ) binding indicating that Mn2+ bound PfApn1 at this site; this was further supported by molecular dynamic simulation. CD spectra analysis further showed requirement of both metal ions for the attainment of PfApn1 β-strand-rich optimal conformation. PfApn1 also functioned as a 3'-phosphatase that would enable removal of 3'-blocks for DNA polymerase activity during BER. Interestingly, unlike Escherichiacoli and yeast EndoIV homologs, PfApn1 lacked 3'-5' exonuclease activity and also did not cleave damaged bases by nucleotide incision repair (NIR). Uncoupling of endonuclease/phosphatase and exonuclease/NIR in PfApn1 suggests that amino acid residues distinct from those critical for endonuclease function are required for exonuclease activity and NIR. Characterization of a critical mitochondrion-targeted AP endonuclease provides evidence for a functional BER pathway in the parasite organelle.

Exog Displays 5′-Exonuclease Activity on Both ssDNA and ssRNA

Human mitochondrial nuclease, EXOG (hEXOG), is essential for repair of damaged mtDNA. It shares sequence homology with the apoptotic endonuclease EndoG. However, apart from being endonuclease, hEXOG also possesses 5′-exonuclease activity which is necessary for the 5′-end damage removal and thus robust DNA repair in human mitochondria. Recently the X-ray crystal structures of hEXOG in apo and dsDNA-bound forms were solved. It was shown that a Wing domain of hEXOG confers a specific 5′-dsDNA exonuclease activity which provides an optimal substrate for subsequent gap-filling and strand ligation steps, thus efficient base-excision repair (BER). Still, the molecular mechanism underlying mitochondrial BER has yet to be clearly defined. Here we report structural and biochemical evidence of 5′-exonuclease activity of hEXOG on ssDNA as well as ssRNA. Our results demonstrate that hEXOG acts as a multifunctional enzyme which may contribute not only to the 5′-end damage removal in mitochondrial BER but also to elimination of unnecessary single-stranded DNA and RNA molecules that may arise during nucleic acid processing in mitochondria.

Enhancing Base Excision Repair of Mitochondrial DNA to Reduce Ischemic Injury Following Reperfusion

We hypothesize that enhancing mitochondrial base excision repair (BER) capability in brain will reduce reperfusion-associated ischemic brain injury. Post-stroke reperfusion was modeled in mice via transient filament occlusion of the middle cerebral artery (60 min) (transient MCAO). Administration of a TAT-modified form of a DNA glycosylase (EndoIII) following reperfusion of the brain reduced resultant brain infarct volume. Protection was dose-dependent, BER enzyme specific, and regionally specific (more effective via the jugular vein). EndoIII is compatible with tissue plasminogen activator (tPA). The time window of a single dose of EndoIII effect is 3 h following reperfusion onset. These data suggest a novel approach to enhance protection of reperfused brain in the setting of revascularization procedures (thrombectomy or thrombolytic therapy) following stroke.

Short-term high temperature treatment reduces viability and inhibits respiration and DNA repair enzymes in Araucaria angustifolia cells.

We evaluated the effect of global warming on Araucaria angustifolia (Bert.) O. Kuntze, a critically endangered native tree of Southern Brazil, by studying the effects of short-term high temperature treatment on cell viability, respiration and DNA repair of embryogenic cells. Compared with control cells grown at 25°C, cell viability was reduced by 40% after incubation at 30 and 37°C for 24 and 6 h, respectively, while 2 h at 40 and 42°C killed 95% of the cells. Cell respiration was unaffected at 30-37°C, but dramatically reduced after 2 h at 42°C. The in vitro activity of enzymes of the base excision repair (BER) pathway was determined. Apurinic/apyrimidine endonuclease, measured in extracts from cells incubated for 2 h at 42°C, was completely inactivated while lower temperatures had no effect. The activities of three enzymes of the mitochondrial BER pathway were measured after 30-min preincubation of isolated mitochondria at 25-40°C and one of them, uracil glycosylase, was completely inhibited at 40°C. We conclude that cell viability, respiration and DNA repair have different temperature sensitivities between 25 and 37°C, and that they are all very sensitive to 40 or 42°C. Thus, A. angustifolia will likely be vulnerable to the short-term high temperature events associated with global warming.

DNA repair in plant mitochondria-a complete base excision repair pathway in potato tuber mitochondria.

Mitochondria are one of the major sites of reactive oxygen species (ROS) production in the plant cell. ROS can damage DNA, and this damage is in many organisms mainly repaired by the base excision repair (BER) pathway. We know very little about DNA repair in plants especially in the mitochondria. Combining proteomics, bioinformatics, western blot and enzyme assays, we here demonstrate that the complete BER pathway is found in mitochondria isolated from potato (Solanum tuberosum) tubers. The enzyme activities of three DNA glycosylases and an apurinic/apyrimidinic (AP) endonuclease (APE) were characterized with respect to Mg2+ dependence and, in the case of the APE, temperature sensitivity. Evidence for the presence of the DNA polymerase and the DNA ligase, which complete the repair pathway by replacing the excised base and closing the gap, was also obtained. We tested the effect of oxidative stress on the mitochondrial BER pathway by incubating potato tubers under hypoxia. Protein carbonylation increased significantly in hypoxic tuber mitochondria indicative of increased oxidative stress. The activity of two BER enzymes increased significantly in response to this oxidative stress consistent with the role of the BER pathway in the repair of oxidative damage to mitochondrial DNA.

Base-excision repair deficiency alone or combined with increased oxidative stress does not increase mtDNA point mutations in mice.

Mitochondrial DNA (mtDNA) mutations become more prevalent with age and are postulated to contribute to the ageing process. Point mutations of mtDNA have been suggested to originate from two main sources, i.e. replicative errors and oxidative damage, but the contribution of each of these processes is much discussed. To elucidate the origin of mtDNA mutations, we measured point mutation load in mice with deficient mitochondrial base-excision repair (BER) caused by knockout alleles preventing mitochondrial import of the DNA repair glycosylases OGG1 and MUTYH (Ogg1 dMTS, Mutyh dMTS). Surprisingly, we detected no increase in the mtDNA mutation load in old Ogg1 dMTS mice. As DNA repair is especially important in the germ line, we bred the BER deficient mice for five consecutive generations but found no increase in the mtDNA mutation load in these maternal lineages. To increase reactive oxygen species (ROS) levels and oxidative damage, we bred the Ogg1 dMTS mice with tissue specific Sod2 knockout mice. Although increased superoxide levels caused a plethora of changes in mitochondrial function, we did not detect any changes in the mutation load of mtDNA or mtRNA. Our results show that the importance of oxidative damage as a contributor of mtDNA mutations should be re-evaluated.

Knockout of Mpv17-Like Protein (M-LPH) Gene in Human Hepatoma Cells Results in Impairment of mtDNA Integrity through Reduction of TFAM, OGG1, and LIG3 at the Protein Levels.

Human Mpv17-like protein (M-LPH) has been suggested to participate in prevention of mitochondrial dysfunction caused by mitochondrial DNA (mtDNA) damage. To clarify the molecular mechanism of M-LPH function, we knocked out M-LPH in human hepatoma HepG2 using CRISPR-Cas9 technology. An increase in mtDNA damage in M-LPH-KO HepG2 cells was demonstrated by PCR-based quantitation and 8-hydroxy-2′-deoxyguanosine (8-OHdG) measurement. Furthermore, confocal immunofluorescence analysis and Western blot analysis of mitochondrial extracts demonstrated that M-LPH-KO caused reductions in the protein levels of mitochondrial transcription factor A (TFAM), an essential factor for transcription and maintenance of mtDNA, and two DNA repair enzymes, 8-oxoguanine DNA glycosylase (OGG1) and DNA ligase 3 (LIG3), both involved in mitochondrial base excision repair (BER). Accordingly, it was suggested that the increase in mtDNA damage was due to a cumulative effect of mtDNA instability resulting from deficiencies of TFAM and diminished ability for BER arising from deficiencies in BER-related enzymes. These findings suggest that M-LPH could be involved in the maintenance of mtDNA, and therefore mitochondrial function, by protecting proteins essential for mtDNA stability and maintenance, in an integrated manner.

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.

Hydrogen peroxide mediated mitochondrial UNG1-PRDX3 interaction and UNG1 degradation

Isoform 1 of uracil-DNA glycosylase (UNG1) is the major protein for initiating base-excision repair in mitochondria and is in close proximity to the respiratory chain that generates reactive oxygen species (ROS). Effects of ROS on the stability of UNG1 have not been well characterized. In the present study, we found that overexpression of UNG1 enhanced cells’ resistance to oxidative stress and protected mitochondrial DNA (mtDNA) from oxidation. Proteomics analysis showed that UNG1 bound to eight proteins in the mitochondria, including PAPSS2, CD70 antigen, and AGR2 under normal growth conditions, whereas UNG1 mainly bound to Peroxiredoxin 3 (PRDX3) via a disulfide linkage under oxidative stress. We further demonstrated that the UNG1-PRDX3 interaction protected UNG1 from ROS-mediated degradation and prevented mtDNA oxidation. Moreover, our results show that ROS-mediated UNG1 degradation was Lon protease 1 (LonP1)-dependent and mitochondrial UNG1 degradation was aggravated by knockdown of PRDX3 expression. Taken together, these results reveal a novel function of UNG1 in the recruitment of PRDX3 to mtDNA under oxidative stress, enabling protection of UNG1 and UNG1-bound DNA from ROS damage and enhancing cell resistance to oxidative stress.

Mitochondrial DNA damage and repair during ischemia–reperfusion injury of the heart

Ischemia–reperfusion (IR) injury of the heart generates reactive oxygen species that oxidize macromolecules including mitochondrial DNA (mtDNA). The 8-oxoguanine DNA glycosylase (OGG1) works synergistically with MutY DNA glycosylase (MYH) to maintain mtDNA integrity. Our objective was to study the functional outcome of lacking the repair enzymes OGG1 and MYH after myocardial IR and we hypothesized that OGG1 and MYH are important enzymes to preserve mtDNA and heart function after IR. Ex vivo global ischemia for 30min followed by 10min of reperfusion induced mtDNA damage that was removed within 60min of reperfusion in wild-type mice. After 60min of reperfusion the ogg1−/− mice demonstrated increased mtDNA copy number and decreased mtDNA damage removal suggesting that OGG1 is responsible for removal of IR-induced mtDNA damage and copy number regulation. mtDNA damage was not detected in the ogg1−/−/myh−/−, inferring that adenine opposite 8-oxoguanine is an abundant mtDNA lesion upon IR. The level and integrity of mtDNA were restored in all genotypes after 35min of regional ischemia and six week reperfusion with no change in cardiac function. No consistent upregulation of other mitochondrial base excision repair enzymes in any of our knockout models was found. Thus repair of mtDNA oxidative base lesions may not be important for maintenance of cardiac function during IR injury in vivo. This article is part of a Special Issue entitled "Mitochondria: From Basic Mitochondrial Biology to Cardiovascular Disease."