DNA and RNA binding by the mitochondrial lon protease is regulated by nucleotide and protein substrate

The ATP-dependent Lon protease belongs to a unique group of proteases that bind DNA. Eukaryotic Lon is a homo-oligomeric ring-shaped complex localized to the mitochondrial matrix. In vitro, human Lon binds specifically to a single-stranded GT-rich DNA sequence overlapping the light strand promoter of human mitochondrial DNA (mtDNA). We demonstrate that Lon binds GT-rich DNA sequences found throughout the heavy strand of mtDNA and that it also interacts specifically with GU-rich RNA. ATP inhibits the binding of Lon to DNA or RNA, whereas the presence of protein substrate increases the DNA binding affinity of Lon 3.5-fold. We show that nucleotide inhibition and protein substrate stimulation coordinately regulate DNA binding. In contrast to the wild type enzyme, a Lon mutant lacking both ATPase and protease activity binds nucleic acid; however, protein substrate fails to stimulate binding. These results suggest that conformational changes in the Lon holoenzyme induced by nucleotide and protein substrate modulate the binding affinity for single-stranded mtDNA and RNA in vivo. Co-immunoprecipitation experiments show that Lon interacts with mtDNA polymerase gamma and the Twinkle helicase, which are components of mitochondrial nucleoids. Taken together, these results suggest that Lon participates directly in the metabolism of mtDNA.     

Thermodynamic characterization of specific interactions between the human Lon protease and G-quartet DNA

Lon is an ATP-powered protease that binds DNA. However, the function of DNA binding by Lon remains elusive. Studies suggest that human Lon (hLon) binds preferentially to a G-rich single-stranded DNA (ssDNA) sequence overlapping the light strand promoter of mitochondrial DNA. This sequence is contained within a 24-base oligonucleotide referred to as LSPas. Here, we use biochemical and biophysical approaches to elucidate the structural properties of ssDNAs bound by hLon, as well as the thermodynamics of DNA binding by hLon. Electrophoretic mobility shift assay and circular dichroism show that ssDNAs with a propensity for forming parallel G-quartets are specifically bound by hLon. Isothermal titration calorimetry demonstrates that hLon binding to LSPas is primarily driven by enthalpy change associated with a significant reduction in heat capacity. Differential scanning calorimetry pinpoints an excess heat capacity upon hLon binding to LSPas. By contrast, hLon binding to an 8-base G-rich core sequence is entropically driven with a relatively negligible change in heat capacity. A considerable enhancement of thermal stability accompanies hLon binding to LSPas as compared to the G-rich core. Taken together, these data support the notion that hLon binds G-quartets through rigid-body binding and that binding to LSPas is coupled with structural adaptation.     

In Vivo Occupancy of Mitochondrial Single-Stranded DNA Binding Protein Supports the Strand Displacement Mode of DNA Replication

Mitochondrial DNA (mtDNA) encodes for proteins required for oxidative phosphorylation, and mutations affecting the genome have been linked to a number of diseases as well as the natural ageing process in mammals. Human mtDNA is replicated by a molecular machinery that is distinct from the nuclear replisome, but there is still no consensus on the exact mode of mtDNA replication. We here demonstrate that the mitochondrial single-stranded DNA binding protein (mtSSB) directs origin specific initiation of mtDNA replication. MtSSB covers the parental heavy strand, which is displaced during mtDNA replication. MtSSB blocks primer synthesis on the displaced strand and restricts initiation of light-strand mtDNA synthesis to the specific origin of light-strand DNA synthesis (OriL). The in vivo occupancy profile of mtSSB displays a distinct pattern, with the highest levels of mtSSB close to the mitochondrial control region and with a gradual decline towards OriL. The pattern correlates with the replication products expected for the strand displacement mode of mtDNA synthesis, lending strong in vivo support for this debated model for mitochondrial DNA replication.     

Lon upregulation contributes to cisplatin resistance by triggering NCLX-mediated mitochondrial Ca2+ release in cancer cells

Mitochondria are the major organelles in sensing cellular stress and inducing the response for cell survival. Mitochondrial Lon has been identified as an important stress protein involved in regulating proliferation, metastasis, and apoptosis in cancer cells. However, the mechanism of retrograde signaling by Lon on mitochondrial DNA (mtDNA) damage remains to be elucidated. Here we report the role of Lon in the response to cisplatin-induced mtDNA damage and oxidative stress, which confers cancer cells on cisplatin resistance via modulating calcium levels in mitochondria and cytosol. First, we found that cisplatin treatment on oral cancer cells caused oxidative damage of mtDNA and induced Lon expression. Lon overexpression in cancer cells decreased while Lon knockdown sensitized the cytotoxicity towards cisplatin treatment. We further identified that cisplatin-induced Lon activates the PYK2-SRC-STAT3 pathway to stimulate Bcl-2 and IL-6 expression, leading to the cytotoxicity resistance to cisplatin. Intriguingly, we found that activation of this pathway is through an increase of intracellular calcium (Ca²⁺) via NCLX, a mitochondrial Na⁺/Ca²⁺ exchanger. We then verified that NCLX expression is dependent on Lon levels; Lon interacts with and activates NCLX activity. NCLX inhibition increased the level of mitochondrial calcium and sensitized the cytotoxicity to cisplatin in vitro and in vivo. In summary, mitochondrial Lon-induced cisplatin resistance is mediated by calcium release into cytosol through NCLX, which activates calcium-dependent PYK2-SRC-STAT3-IL-6 pathway. Thus, our work uncovers the novel retrograde signaling by mitochondrial Lon on resistance to cisplatin-induced mtDNA stress, indicating the potential use of Lon and NCLX inhibitors for better clinical outcomes in chemoresistant cancer patients.Subject terms: Cancer therapeutic resistance, Mitochondria, Calcium and vitamin D     

The DNA glycosylase Ogg1 defends against oxidant-induced mtDNA damage and apoptosis in pulmonary artery endothelial cells

Emerging evidence suggests that mitochondrial (mt) DNA damage may be a trigger for apoptosis in oxidant-challenged pulmonary artery endothelial cells (PAECs). Understanding the rate-limiting determinants of mtDNA repair may point to new targets for intervention in acute lung injury. The base excision repair (BER) pathway is the only pathway for oxidative damage repair in mtDNA. One of the key BER enzymes is Ogg1, which excises the base oxidation product 8-oxoguanine. Previously we demonstrated that overexpression of mitochondrially targeted Ogg1 in PAECs attenuated apoptosis induced by xanthine oxidase (XO) treatment. To test the idea that Ogg1 is a potentially rate-limiting BER determinant protecting cells from oxidant-mediated death, PAECs transfected with siRNA to Ogg1 were challenged with XO and the extent of mitochondrial and nuclear DNA damage was determined along with indices of apoptosis. Transfected cells demonstrated significantly reduced Ogg1 activity, which was accompanied by delayed repair of XO-induced mtDNA damage and linked to increased XO-mediated apoptosis. The nuclear genome was undamaged by XO in either control PAECs or cells depleted of Ogg1. These observations suggest that Ogg1 plays a critical and possibly rate-limiting role in defending PAECs from oxidant-induced apoptosis by limiting the persistence of oxidative damage in the mitochondrial genome.     

The expression of Exonuclease III from E. coli in mitochondria of breast cancer cells diminishes mitochondrial DNA repair capacity and cell survival after oxidative stress

The ability to sensitize cancer cells to radiation would be highly beneficial for successful cancer treatment. One mode of action for ionizing radiation is the induction of cell death through infliction of extensive oxidative damage to cellular DNA, including mitochondrial DNA (mtDNA). The ability of cells to repair mtDNA and otherwise maintain the integrity of their mitochondria is vital for protection of the cells against oxidative damage. Because efficient repair of oxidative damage in mtDNA may play a crucial role in cancer cell resistance, interference with this repair process could be an effective way to achieve a radiation sensitive phenotype in otherwise resistant cancer cells. Successful repair of DNA is achieved through a precise and highly regulated multistep process. Expression of excessive amounts of one of the repair enzymes may cause an imbalance of the whole repair system and lead to the loss of repair efficiency. To study the effects of changing mtDNA repair capacity on overall cell survival following oxidative stress, we expressed a bacterial repair enzyme, Exonuclease III (ExoIII) containing the mitochondrial targeting signal of manganese superoxide dismutase, in a human malignant breast epithelial cell line, MDA-MB-231. Following transfection, specific exonuclease activity was found in mitochondrial extracts. In order to examine the effects on repair of oxidative damage in mtDNA, cells were exposed to the enzyme xanthine oxidase and its substrate hypoxanthine. mtDNA repair was evaluated using quantitative Southern blot analysis. The results revealed that cells expressing ExoIII in mitochondria are deficient in mtDNA repair when compared with control cells that express ExoIII without MTS. This diminished mtDNA repair capacity rendered MDA-MB-231 cells more sensitive to oxidative damage, which resulted in a decrease in their long-term survival following oxidative stress.     

The role of mitochondrial DNA damage in the citotoxicity of reactive oxygen species

Mitochondria contain their own genome, a small circular molecule of around 16.5 kbases. The mitochondrial DNA (mtDNA) encodes for only 13 polypeptides, but its integrity is essential for mitochondrial function, as all 13 proteins are regulatory subunits of the oxidative phosphorylation complexes. Nonetheless, the mtDNA is physically associated with the inner mitochondrial membrane, where the majority of the cellular reactive oxygen species are generated. In fact, the mitochondrial DNA accumulates high levels of oxidized lesions, which have been associated with several pathological and degenerative processes. The cellular responses to nuclear DNA damage have been extensively studied, but so far little is known about the functional outcome and cellular responses to mtDNA damage. In this review we will discuss the mechanisms that lead to damage accumulation and the in vitro models we are establishing to dissect the cellular responses to oxidative damage in the mtDNA and to sort out the differential cellular consequences of accumulation of damage in each cellular genome, the nuclear and the mitochondrial genome.     

Mitochondrial genome maintenance in health and disease

Human mitochondria harbor an essential, high copy number, 16,569 base pair, circular DNA genome that encodes 13 gene products required for electron transport and oxidative phosphorylation. Mutation of this genome can compromise cellular respiration, ultimately resulting in a variety of progressive metabolic diseases collectively known as ‘mitochondrial diseases’. Mutagenesis of mtDNA and the persistence of mtDNA mutations in cells and tissues is a complex topic, involving the interplay of DNA replication, DNA damage and repair, purifying selection, organelle dynamics, mitophagy, and aging. We briefly review these general elements that affect maintenance of mtDNA, and we focus on nuclear genes encoding the mtDNA replication machinery that can perturb the genetic integrity of the mitochondrial genome.     

Oxidative DNA damage causes mitochondrial genomic instability in Saccharomyces cerevisiae

Mitochondria contain their own genome, the integrity of which is required for normal cellular energy metabolism. Reactive oxygen species (ROS) produced by normal mitochondrial respiration can damage cellular macromolecules, including mitochondrial DNA (mtDNA), and have been implicated in degenerative diseases, cancer, and aging. We developed strategies to elevate mitochondrial oxidative stress by exposure to antimycin and H(2)O(2) or utilizing mutants lacking mitochondrial superoxide dismutase (sod2Delta). Experiments were conducted with strains compromised in mitochondrial base excision repair (ntg1Delta) and oxidative damage resistance (pif1Delta) in order to delineate the relationship between these pathways. We observed enhanced ROS production, resulting in a direct increase in oxidative mtDNA damage and mutagenesis. Repair-deficient mutants exposed to oxidative stress conditions exhibited profound genomic instability. Elimination of Ntg1p and Pif1p resulted in a synergistic corruption of respiratory competency upon exposure to antimycin and H(2)O(2). Mitochondrial genomic integrity was substantially compromised in ntg1Delta pif1Delta sod2Delta strains, since these cells exhibit a total loss of mtDNA. A stable respiration-defective strain, possessing a normal complement of mtDNA damage resistance pathways, exhibited a complete loss of mtDNA upon exposure to antimycin and H(2)O(2). This loss was preventable by Sod2p overexpression. These results provide direct evidence that oxidative mtDNA damage can be a major contributor to mitochondrial genomic instability and demonstrate cooperation of Ntg1p and Pif1p to resist the introduction of lesions into the mitochondrial genome.     

Repair of oxidative damage in mitochondrial DNA of Saccharomyces cerevisiae: involvement of the MSH1-dependent pathway

Mitochondrial DNA (mtDNA) is located close to the respiratory chain, a major source of reactive oxygen species (ROS). This proximity makes mtDNA more vulnerable than nuclear DNA to damage by ROS. Therefore, the efficient repair of oxidative lesions in mtDNA is essential for maintaining the stability of the mitochondrial genome. A series of genetic and biochemical studies has indicated that eukaryotic cells, including the model organism Saccharomyces cerevisiae, use several alternative strategies to prevent mutagenesis induced by endogenous oxidative damage to nuclear DNA. However, apart from base excision repair (BER), no other pathways involved in the repair of oxidative damage in mtDNA have been identified. In this study, we have examined mitochondrial mutagenesis in S. cerevisiae cells which lack the activity of the Ogg1 glycosylase, an enzyme playing a crucial role in the removal of 8-oxoG, the most abundant oxidative lesion of DNA. We show that the overall frequency of the mitochondrial oligomycin-resistant (Olir) mutants is increased in the ogg1 strain by about one order of magnitude compared to that of the wild-type strain. Noteworthy, in the mitochondrial oli1 gene, G:C to T:A transversions are generated approximately 50-fold more frequently in the ogg1 mutant relative to the wild-type strain. We also demonstrate that the increased frequency of Olir mutants in the ogg1 strain is markedly reduced by the presence of plasmids encoding Msh1p, a homologue of the bacterial mismatch protein MutS, which specifically functions in mitochondria. This suppression of the mitochondrial mutator phenotype of the ogg1 strain seems to be specific, since overexpression of the mutant allele msh1-R813W failed to exert this effect. Finally, we also show that the increased frequency of Olir mutants arising in an msh1/MSH1 heterozygote grown in glucose-containing medium is further enhanced if the cells are cultivated in glycerol-containing medium, i.e. under conditions when the respiratory chain is fully active. Taken together, these results strongly suggest that MSH1-dependent repair represents a significant back-up to mtBER in the repair of oxidative damage in mtDNA.     

Changes in the human mitochondrial genome after treatment of malignant disease

Mitochondrial DNA (mtDNA) is the only extrachromosomal DNA in human cells. The mitochondrial genome encodes essential information for the synthesis of the mitochondrial respiratory chain. Inherited defects of this genome are an important cause of human disease. In addition, the mitochondrial genome seems to be particularly prone to DNA damage and acquired mutations may have a role in ageing, cancer and neurodegeneration. We wished to determine if radiotherapy and chemotherapy used in the treatment of cancer could induce changes in the mitochondrial genome. Such changes would be an important genetic marker of DNA damage and may explain some of the adverse effects of treatment. We studied samples from patients who had received radiotherapy and chemotherapy for point mutations within the mtDNA control region, and for large-scale deletions. In blood samples from patients, we found a significantly increased number of point mutations compared to the control subjects. In muscle biopsies from 7 of 8 patients whom had received whole body irradiation as well as chemotherapy, the level of a specific mtDNA deletion was significantly greater than in control subjects. Our studies have shown that in patients who have been treated for cancer there is an increased level of mtDNA damage.     

Reduced stimulation of recombinant DNA polymerase γ and mitochondrial DNA (mtDNA) helicase by variants of mitochondrial single-stranded DNA-binding protein (mtSSB) correlates with defects in mtDNA replication in animal cells

The mitochondrial single-stranded DNA-binding protein (mtSSB) is believed to coordinate the functions of DNA polymerase γ (pol γ) and the mitochondrial DNA (mtDNA) helicase at the mtDNA replication fork. We generated five variants of the human mtSSB bearing mutations in amino acid residues specific to metazoans that map on the protein surface, removed from the single-stranded DNA (ssDNA) binding groove. Although the mtSSB variants bound ssDNA with only slightly different affinities, they exhibited distinct capacities to stimulate the DNA polymerase activity of human pol γ and the DNA unwinding activity of human mtDNA helicase in vitro. Interestingly, we observed that the variants with defects in stimulating pol γ had unaltered capacities to stimulate the mtDNA helicase; at the same time, variants showing reduced stimulation of the mtDNA helicase activity promoted DNA synthesis by pol γ similarly to the wild-type mtSSB. The overexpression of the equivalent variants of Drosophila melanogaster mtSSB in S2 cells in culture caused mtDNA depletion under conditions of mitochondrial homeostasis. Furthermore, we observed more severe reduction of mtDNA copy number upon expression of these proteins during recovery from treatment with ethidium bromide, when mtDNA replication is stimulated in vivo. Our findings suggest that mtSSB uses distinct structural elements to interact functionally with its mtDNA replisome partners and to promote proper mtDNA replication in animal cells.     

Minisatellite binding protein Msbp-1 is a sequence-specific single-stranded DNA-binding protein.

Msbp-1 is a minisatellite-specific DNA-binding protein. Using synthetic binding substrates, we now show that Msbp-1 binds not to double-stranded DNA, but exclusively to single-stranded DNA. Binding is specific to the guanine-rich strand of the minisatellite duplex, interactions with the cytosine-rich strand being undetectable by southwestern analysis. Furthermore, the binding site required for successful DNA-protein interactions appears to be two or more minisatellite repeat units. We have also isolated, by whole-genome PCR and cloning, one Msbp-1 binding site from the human genome. Again, the binding strand of this molecule contains a repetitive G-rich structure equivalent to that of a small minisatellite. These observations are discussed with respect to other single-stranded DNA-binding proteins known to play a role in recombination processes.     

Interaction of Sirt3 with OGG1 contributes to repair of mitochondrial DNA and protects from apoptotic cell death under oxidative stress

Sirtuin 3 (Sirt3), a major mitochondrial NAD⁺-dependent deacetylase, targets various mitochondrial proteins for lysine deacetylation and regulates important cellular functions such as energy metabolism, aging, and stress response. In this study, we identified the human 8-oxoguanine-DNA glycosylase 1 (OGG1), a DNA repair enzyme that excises 7,8-dihydro-8-oxoguanine (8-oxoG) from damaged genome, as a new target protein for Sirt3. We found that Sirt3 physically associated with OGG1 and deacetylated this DNA glycosylase and that deacetylation by Sirt3 prevented the degradation of the OGG1 protein and controlled its incision activity. We further showed that regulation of the acetylation and turnover of OGG1 by Sirt3 played a critical role in repairing mitochondrial DNA (mtDNA) damage, protecting mitochondrial integrity, and preventing apoptotic cell death under oxidative stress. We observed that following ionizing radiation, human tumor cells with silencing of Sirt3 expression exhibited deteriorated oxidative damage of mtDNA, as measured by the accumulation of 8-oxoG and 4977 common deletion, and showed more severe mitochondrial dysfunction and underwent greater apoptosis in comparison with the cells without silencing of Sirt3 expression. The results reported here not only reveal a new function and mechanism for Sirt3 in defending the mitochondrial genome against oxidative damage and protecting from the genotoxic stress-induced apoptotic cell death but also provide evidence supporting a new mtDNA repair pathway.     

Mgm101p is a novel component of the mitochondrial nucleoid that binds DNA and is required for the repair of oxidatively damaged mitochondrial DNA

Maintenance of mitochondrial DNA (mtDNA) during cell division is required for progeny to be respiratory competent. Maintenance involves the replication, repair, assembly, segregation, and partitioning of the mitochondrial nucleoid. MGM101 has been identified as a gene essential for mtDNA maintenance in S. cerevisiae, but its role is unknown. Using liquid chromatography coupled with tandem mass spectrometry, we identified Mgm101p as a component of highly enriched nucleoids, suggesting that it plays a nucleoid-specific role in maintenance. Subcellular fractionation, indirect immunofluorescence and GFP tagging show that Mgm101p is exclusively associated with the mitochondrial nucleoid structure in cells. Furthermore, DNA affinity chromatography of nucleoid extracts indicates that Mgm101p binds to DNA, suggesting that its nucleoid localization is in part due to this activity. Phenotypic analysis of cells containing a temperature sensitive mgm101 allele suggests that Mgm101p is not involved in mtDNA packaging, segregation, partitioning or required for ongoing mtDNA replication. We examined Mgm101p's role in mtDNA repair. As compared with wild-type cells, mgm101 cells were more sensitive to mtDNA damage induced by UV irradiation and were hypersensitive to mtDNA damage induced by gamma rays and H2O2 treatment. Thus, we propose that Mgm101p performs an essential function in the repair of oxidatively damaged mtDNA that is required for the maintenance of the mitochondrial genome.     

Replicating animal mitochondrial DNA

The field of mitochondrial DNA (mtDNA) replication has been experiencing incredible progress in recent years, and yet little is certain about the mechanism(s) used by animal cells to replicate this plasmid-like genome. The long-standing strand-displacement model of mammalian mtDNA replication (for which single-stranded DNA intermediates are a hallmark) has been intensively challenged by a new set of data, which suggests that replication proceeds via coupled leading- and lagging-strand synthesis (resembling bacterial genome replication) and/or via long stretches of RNA intermediates laid on the mtDNA lagging-strand (the so called RITOLS). The set of proteins required for mtDNA replication is small and includes the catalytic and accessory subunits of DNA polymerase γ, the mtDNA helicase Twinkle, the mitochondrial single-stranded DNA-binding protein, and the mitochondrial RNA polymerase (which most likely functions as the mtDNA primase). Mutations in the genes coding for the first three proteins are associated with human diseases and premature aging, justifying the research interest in the genetic, biochemical and structural properties of the mtDNA replication machinery. Here we summarize these properties and discuss the current models of mtDNA replication in animal cells.