Mitochondrial Dna Damage And Repair

mtDNA mutation rate is faster than alterations in nuclear DNA

In 1979, it was estimated that nucleotide substitutions are present at a 10-fold higher frequency in mtDNA than in nuclear DNA. The rate of mtDNA evolution is faster than that of nuclear DNA (13,14), which may reflect an increased susceptibility to mutations (15). The mtDNA undergoes a continuous turnover both in mitotic and post-mitotic cells that increases the chance of mutations. Alterations to the structure of mtDNA during aging in the form of circular dimers and small deletions/insertions had first been identified in a variety of tissues in rodents (16) and later in almost all human tissues, e.g. post-mitotic differentiated tissues such as skeletal muscle, heart muscle, and brain (17-20). Mutations in highly replicative tissues such as skin (21) (or blood (22)) were also found but seem to depend on additional factors.

The consequences of this increased mutation rate have been of great interest since the discovery of specific mtDNA mutations that are likely involved in aging or several degenerative diseases (in (2,3)). The higher mutation rate of mtDNA can be caused by two general factors: an increased susceptibility of mtDNA to mutations and a relatively insufficient repertoire of enzymatic DNA repair.

Damage to mtDNA is higher than to nuclear DNA

It was a relatively early observation that, compared to nuclear DNA, mtDNA contains an increased level of damage, i.e. 7-hydroxo-8-oxo-deoxyguanosine (8-oxo-dG) (23,24). In 1997, Yakes and van Houten (25) evaluated the formation and repair of H2O2-induced DNA damage in a 16.2-kb mitochondrial and a 17.7-kb P-Globin gene by using a quantitative PCR (QPCR). This experiment is based on the fact that H2O2-induced DNA lesions, including oxidative damage such as strand breaks, base modifications, and AP sites, will block the progression of the polymerase resulting in a decrease in amplification of the target sequence: only DNA templates that do not contain these lesions will be amplified. After the treatment of human fibroblasts with 200-^M H2O2 for 60 min, damage to mtDNA occurred more rapidly than to the nuclear fragment. DNA repair in the nuclear DNA occurred within 1.5 h but no repair was observed in mtDNA even after 3.5 h. However, reduction of the exposure to 15 min resulted in mtDNA repair within 1.5 h with the same efficiency as in the nuclear DNA (25). This was one of the first reports showing that mitochondria were proficient in the repair of H2O2-induced DNA damage following short exposures, and that longer treatment lead to persistent mtDNA damage, which is additionally higher than in nuclear DNA. In the same year, Salazar and van Houten confirmed these results by comparing repair of oxidative DNA damage in the nucleus and mitochondria of human fibroblasts after exposure to glucose oxidase (GO), which is excellent for generating a steady concentration of H2O2. They found 5-7 times higher damage to mtDNA than to any of the investigated nuclear DNA loci (26). The fact that mtDNA displayed an increased susceptibility to radiation-induced loss of integrity compared to nuclear DNA was confirmed by other authors in the last few years (27). Using a dissociation-enhanced lanthanic fluoroimmunoassay (DELFIA), Olivero et al. demonstrated that cisplatin-DNA adducts form in mtDNA in a preferential manner compared to nuclear DNA (28). Others showed that ultraviolet radiation C (UVC) exposure induced significantly higher amounts of a special DNA alteration (pyrimidine[6-4] pyrim-idone photoproducts (6-4 PPs)) in human mtDNA whereas cyclobutane pyrimidine dimers (CPDs) occurred in similar frequencies (29).

Mitochondrial DNA is extremely vulnerable

Mitochondria are semi-autonomous organelles. Their main function is to generate ATP during OXPHOS (see (30) for review). They cover more than 80% of the energy needs of the cell and contain the only DNA outside the nucleus in mammalian cells. Each mitochondrion contains up to 10 copies of its genome, whereas each cell contains approximately 104 mitochondria. The mitochondrial genome (mtDNA: 16,569 bp) encodes 13 polypeptides involved in OXPHOS, 22 tRNAs, and 2 ribosomal RNAs. The mito-chondrial structures are very susceptible to oxidative stress as shown by many reports detecting lipid peroxidation (31), protein oxidation (32), and mtDNA alterations (33). The mitochondrial genome appears to be especially sensitive to endogenous and environmental mutagens (34) since the molecule is located at the matrix surface of the inner membrane, where it is close to the major source of reactive oxygen species (ROS) produced by the respiratory chain; moreover lacking introns and being devoid of histones (33) and other protecting DNA-associated proteins. The mtDNA is found to contain approximately 10-fold higher levels of oxidized bases than nuclear DNA (35).

Mitochondria also have a matrix-side negative membrane potential for OXPHOS. This membrane potential concentrates lipophilic cations inside mitochondria up to approximately 1000-fold, and some therapeutic reagents are lipophilic cations and are thus prone to damage mitochondria (36). In addition, mtDNA is exposed to all other mutagenic forces to which all DNA molecules are subjected. To make it more difficult, mtDNA is also attacked by exposure to certain chemical, chemotherapeutic, and antiviral agents (37,38), whereas the mitochondrial genome seems to be, additionally, the preferred target of some known toxins and carcinogens (39,40). The formation of complexes between certain substances and mtDNA may be favored by the closed circular structure of the mitochondrial genome (41), and probably involves intercalation of the foreign chemicals in the DNA. The high intrinsic mutability of the mitochondrial genome is reflected in an insertion error rate of mtDNA polymerase y of 1/7000, which may lead to nucleotide substitutions and deletions.

ROS are produced continuously in the mitochondria

ROS are produced continuously at a high rate as by-products of aerobic metabolism, including oxygen-free radicals, such as the superoxide radical anion as the primary product of one-electron dioxygen reduction nitric oxide and the derived peroxynitrite, the radical superoxide, singlet oxygen, and the strong non-radical oxidant H2O2, hydroxyl radicals (some are produced by radiation) (42). H2O2 can be reduced to the highly reactive hydroxyl radical OH- by a metal through the Fenton reaction or during exposure to ionizing radiation which can cause additional DNA damage. It induces at least eleven different base products whereas 35-55% of those alterations are expected to result in strong blocks to the polymerase (41). It is calculated that 1-4% of the oxygen reacting with the respiratory chain is incompletely reduced to ROS (24). Since mitochondria are the major producers of ROS, they are particularly susceptible to their attacks. Even ROS produced outside the mitochondria may damage mitochondrial structures as Beyer et al. showed in experiments with isolated hepatocytes (43).

ROS can also be produced by exogenous factors such as chemical agents and UV light, whereas different wavelengths of UV radiation cause different kinds of damage to the DNA and consequently, different kinds of mutations also (reviewed in (44)).

ROS lead to a variety of DNA adducts

When mtDNA is the target, the attack of ROS can lead to single-strand breaks, abasic sites, and possibly more than 100 different types of damaged bases such as thymine glycol (TG) and 7-hydroxo-8-oxo-deoxyguanosine (8-oxo-dG) (reviewed in (45)), one of the most abundant lesions (46) 8-oxo-dG will often adopt the syn conformation, allowing it to mispair with deoxyadenosine which results in G^T transversion when repair is absent (47). Reports show that steady-state levels of 8-oxo-dG are 10-fold higher in mtDNA than in nuclear DNA and increase dramatically with age (33).

The major ultraviolet lesions in DNA are CPDs followed by 6-4 PPs as well as much lower amounts of purine dimers, pyrimidine monoadducts, and a photoproduct between adjacent A and T bases (reviewed in (48)).

Mitochondria contain their own protective substances against ROS

Mitochondria contain antioxidant enzymes, including superoxide dismutase glutathione peroxidase, and lipid-soluble antioxidants such as vitamin E, ubiquinol, and coenzyme Q

(reviewed in (2)). Ubiquinol may exert its antioxidant function indirectly by reducing a-tocopheroxyl radicals back to vitamin E directly as a quencher of oxygen and lipid per-oxyl radicals. Normally, damage to the mtDNA would be disposed of by these enzymes in the mitochondria. However, during aging or in some diseases the activities or quantities of these scavengers are decreased (49) so that an increasing proportion of the ROS and free radicals are not efficiently disposed of and thereby elevate the oxidative stress of the mitochondria (reviewed in (2)). Mitochondria also contain an error avoidance mutT homolog. The mitochondria-specific polymerase, the y-polymerase, readily mis-incorporates 8-oxo-dG opposite adenine. For prevention of such damage, mammalian mitochondria possess mutT homologs, which hydrolyze 8-oxo-dGTP to 8-oxo-dGMP (50).

Mitochondria contain mammalian mtDNA repair enzymes

The isolation of oxidative damage repair enzymes from mitochondria is rather complicated, since these enzymes are usually expressed at very low levels in the cell. In addition, it is important to isolate pure mitochondria in sufficient quantities, and without nuclear contaminations. This is possibly the reason for the slow progress in the discovery of DNA repair enzymes in mitochondria. However, in the last years, several mammalian mtDNA repair enzymes have been detected, including a uracil DNA glycolysase, AP endonucleases, a methyltransferase, a ligase, a DNA polymerase, a pyrimidine hydrate DNA glycosylase (summarized in (51)), as well as a mitochondrial oxidative damage endonuclease (mtODE) which is specific for 8-oxo-dG (52,53).

The first report of the purification of an 8-oxo-dG DNA damage-processing enzyme from mitochondria, the mtODE, was published in 1997 by Croteau et al. (54). They extracted mtODE from rat liver mitochondria and showed that the purified enzyme was able to recognize and incise 8-oxo-dG and abasic sites in duplex PCR (54).

Mitochondria lack nucleotide excision repair (NER)

NER is the most important DNA repair pathway in the cell. It corrects the majority of bulky lesions in nuclear DNA (55,56). Since mtDNA is apparently affected by the same alterations as nuclear DNA, the question arises whether mitochondria contain the same repair mechanisms. In 1974, the first report was published showing that mitochondria are not able to repair CPDs (57). Other authors confirmed these results in the subsequent years (51,58). Other scientists used a restriction site mutation method (RMS) to detect DNA alterations. They analyzed the induction and repair of the two major UV-induced photolesions, CPDs, and 6-4 PPs in mtDNA vs. nuclear DNA of primary human fibroblasts and embryonic kidney 293 cells. In all investigated cells, repair of those UV-induced damages was absent during a 24-h incubation period (29).

Mammalian mitochondria are also unable to repair DNA ICLs induced by psoralen (HMT - 4'-hydroxy-methyl-4,5',8-trimethylpsoralen) as shown in CHO cells by (59). The authors investigated the formation and removal of ICLs after incubation with 75 ng/ml HMT and exposure to UVA light in both mtDNA and nuclear DNA (DFHR gene). These alterations are thought to be repaired by NER and recombinational repair.

Within 24 h, 80% of the cross-links were efficiently removed from the DFHR gene, whereas the mtDNA showed a 7.5-fold higher level of cross-links, which is additionally not repaired in the mitochondrial genome (59).

Despite these findings, mitochondria are able to repair ICLs per se, since another working group demonstrated removal of ICLs induced by the cancer chemotherapeutic drug ds-diamminedichloroplatinum[II] (cisplatin) in mitochondria from CHO cells at a rate comparable to that in the nuclear DNA. They detected a minimal repair of cisplatin-induced intrastrand cross-links and an efficient repair of ICLs as evidenced by 70% of the induced lesions being removed within 24 h. Additionally, the authors showed repair of N-methylpurines following exposure to methylnitrosourea (70% removal by 24 h) (51). However, the assay used in this study measures the conversion of double-stranded cross-linked DNA to single-stranded DNA and therefore reflects the "unhooking" of one arm of the cross-link but not necessarily complete repair of the cross-link. Since mitochondria are deficient of the initial incision steps required for the removal of HMT ICLs, but capable of performing the initial unhooking event required for cisplatin cross-link repair, one could suggest the involvement of different repair pathways for the initial step of the repair of these two types of lesions (59).

Mitochondria are capable of removing various kinds of damage

As a fact, mitochondria cannot repair UV-induced pyrimidine dimers, while these damages are efficiently processed in the nucleus by NER, as mentioned above. The repair of damage caused by cisplatin and nitrogen mustard, agents which are known to induce DNA alterations that are repaired by NER, is inefficient as shown by (5).

Meanwhile, there are several reports showing that mitochondria repair some forms of DNA damage, i.e. induced by UV light, monofunctional alkylation agents, bleomycin, cisplatin, alloxan, streptozotocin, or acridine orange (see Table 1 for overview).

Shen et al. demonstrated the repair of mtDNA damage induced by the naturally occurring, radiometric drug bleomycin (60). Experiments with the human WI-38 cellinie showed more than 80% repair of damaged mtDNA within 2 h, whereas no additional repair of the remaining 20% was observed after 4 h. Summarizing these findings and former results on mtDNA repair of N-methylpurines, ICLs, and a variety of oxidative lesions induced by alloxan with the observed deficiencies in repair of UV-induced CPDs or bulky adducts, the authors suggest that there are at least two different repair mechanisms for mitochondria: a slower repair for lesions such as ICLs, with an approximate repair of 70-80% within 24 h, and a rapid repair of oxidative damage in which everything is repaired within 4 h (60). Oxidative damage, as detected by the FPG protein (E. coli formamidopyrimidine-DNA glycosylase), is repaired in mtDNA from rat cells (61), CHO cells (62) as well as from human fibroblasts (63).

Taffe and coworkers used acridine orange plus visible light to generate oxidative damage, and FPG protein was used in the GSR assay to assess the repair of FPG-sensitive sites (62). The induced damage was repaired from both mtDNA and nuclear DNA. Approximately 65% of the lesions were repaired within 4 h, and the repair in the mtDNA was as fast as in the compared nuclear DHFR gene. Damage of mtDNA induced by alloxan can

Table 1. Repair of mitochondrial DNA induced by different stressors

Inducer

Assay

Damage

Repair

Reference

Acridine orange (+ UV light)

- CHO cells, compare DHFR to mtDNA, lesion recognition by FPG

- Base lesions

- Yes, 84% repair within 4 h, equal to the nuclear DNA

(162)

Alloxan

- Cultured rat cells, quantitative Southern blot

- Variety of oxidative damage to bases and sugar-phosphate backbone

- Yes, 100% by 4 h

(41)

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