Mitochondrial DNA repair pathways

Mutation Research 434 Ž1999. 137–148 www.elsevier.comrlocaterdnarepair Community address: www.elsevier.comrlocatermutres

Section I. Damage and repair in mitochondria

Mitochondrial DNA repair pathways Deborah L. Croteau b, Rob. H. Stierum a , Vilhelm A. Bohr a

a,)

Laboratory of Molecular Genetics, National Institute on Aging, NIH 5600 Nathan Shock Dr., Baltimore, MD 21224, USA b Department of Molecular and Cell Biology, 401 Barker Hall, UniÕersity of California, Berkeley CA 94720, USA Accepted 3 May 1999

Abstract DNA repair mechanisms are fairly well characterized for nuclear DNA while knowledge regarding the repair mechanisms operable in mitochondria is limited. Several lines of evidence suggest that mitochondria contain DNA repair mechanisms. DNA lesions are removed from mtDNA in cells exposed to various chemicals. Protein activities that process damaged DNA have been detected in mitochondria. As will be discussed, there is evidence for base excision repair ŽBER., direct damage reversal, mismatch repair, and recombinational repair mechanisms in mitochondria, while nucleotide excision repair ŽNER., as we know it from nuclear repair, is not present. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Mitochondrial DNA repair pathways; Base excision repair; Nucleotide excision repair

1. Introduction Mammalian mitochondrial DNA ŽmtDNA. exists as a double-stranded closed circular 16.5 kb DNA molecule that is packaged into nucleoid structures within the matrix space of the mitochondrion. MtDNA codes for 13 proteins, 22 tRNA and 2 rRNA species. The majority of the proteins that are encoded by the mtDNA participate in the mitochondrial electron transport chain. Due to the proximity of mtDNA to the electron transport system, a higher steady-state level of oxidative damage has been reported in mtDNA relative to the levels in nuclear DNA w46x. The steady-state level of adducts within the mtDNA is a function of both adduct formation as well as adduct removal, DNA repair, which is the subject of this review. This review will discuss some ) Corresponding author. Tel.: q1-410-558-8162; fax: q1-410558-8157; E-mail: [email protected]

of the recent findings on DNA repair mechanisms in mitochondria but will not discuss the recent controversy over mtDNA adduct level measurements because this has been reviewed recently w5x. The importance of the maintenance of the mitochondrial genome cannot be understated. A small number of mitochondrial DNA mutations and deletions have been attributed to a vast number of mitochondrial myopathy disorders and aging w65,66x. In addition, recent work has shown that there are specific patterns of mutations in mtDNA from human tumors which may have implications for the abnormal metabolic and apoptotic processes in cancers w41x.

2. Base excision repair The BER pathway is initiated by DNA glycosylases, a class of enzymes that recognize a specific set

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of modified bases such as 8-oxodeoxyguanosine Ž8oxodG. or thymine glycol Žfor a comprehensive review on glycosylases, see Ref. w27x.. Glycosylases cleave the N-glycosylic bond between the modified base and the sugar. Simple glycosylases only cleave the N-glycosylic bond while glycosylaserAP lyase enzymes cleave both the N-glycosylic bond and the DNA phosphate backbone. Following the glycosylase step, an apyrimidinicrapurinic endonuclease ŽAP endo. is required to incise the DNA backbone, if it is following the action of a simple glycosylase. If it follows the action of a glycosylaserAP lyase the AP endo must remove the 3X deoxyribose moiety. In either case, the role of the AP endo is to generate a 3X-hydroxyl group, which can be extended by a DNA polymerase. Then the process is completed by a DNA ligase, which joins the free DNA ends Žfor a review, see Ref. w52x.

assay, biochemical assays and PCR based methods. The conclusion is that mitochondria possess efficient repair mechanisms for oxidative DNA damage. Studies on mtDNA damage and repair have traditionally required the purification of mitochondria and mtDNA. As an alternative approach the, gene specific repair assay w6x was modified to detect various DNA lesions in addition to UV induced dimers. Bacterial repair enzymes that recognize and cleave the DNA at specific lesions are used. Oxidative lesions can be detected in the entire mitochondrial genome or in parts of it, and can be compared to the lesions present in the nuclear DNA from the same biological sam ple. Also, strand bias, or transcription-coupled repair ŽTCR., can be assayed with this approach ŽFig. 1.. Furthermore, this proce-

2.1. Repair of uracil The first DNA glycosylase activity identified in mitochondria was uracil DNA glycosylase ŽUDG. w1x. Subsequently, UDG was purified from rat liver and the mitochondrial UDG was shown to have slightly different biochemical properties than the nuclear rat UDG w16,17x. In yeast, mutational inactivation of the nuclear isoform of UDG had no effect upon the mitochondrial UDG activity w8x. Taken together, these results suggested that the mitochondrial and nuclear forms of UDG were derived from two different genes. However, subsequent research revealed that the mitochondrial and nuclear isoforms are in fact derived from the same gene by alternative splicing and utilization of two different start codons w33,34,58x. As will be discussed below, the use of alternative splicing to fuse a mitochondrial localization sequence onto genes which code for DNA repair proteins in order to facilitate mitochondrial localization is becoming a common theme. 2.2. Repair of oxidatiÕe damage BER enzymes in nuclear DNA mediate the repair of oxidative damage. The repair of oxidative damage to mtDNA has been measured using a variety of different methods including the gene-specific repair

Fig. 1. Gene-specific repair assay.

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dure avoids the oxidation of the mtDNA, which may occur during the extraction procedure used in other methods. Using the gene specific repair assay and a variety of DNA damaging agents, repair of strand breaks and alkali sensitive sites has been demonstrated in rodent and human mtDNA Žfor a review, see Ref. w13x.. The formamidopyrimidine Žfapy. DNA glycosylase ŽFpg. enzyme detects 8-oxodG and the ringopened FaPy lesion. It cleaves DNA at the site of the lesion using its associated AP lyase activity. Repair of Fpg-sensitive sites in mtDNA has been reported for rat cells w20x, CHO cells w61x and human cells w2x. In the study by Taffe et al. w61x, acridine orange plus light ŽAOrlight. was used as a method to generate oxidative damage, and the Fpg protein was used to detect the damage. The AOrlight induced DNA damage was repaired from both mt- and nuclear DNA sequences. Approximately 65% of the lesions were repaired within 4 h, and the repair in the mtDNA was as fast or faster as in the nuclear dihydrofolate reductase ŽDHFR. gene, which was also assayed in the experiments. The mechanism of mitochondrial repair was further examined by Anson et al. w2x who assayed the repair of 8-oxodG from human mtDNA using the gene-specific repair assay. Again, the Fpg enzyme was used to detect the lesions. In this case, the cells were exposed to methylene blue plus light before the repair, since this treatment has been shown to be highly specific for the induction of 8-oxodG w7,44,51x. Anson found no strand bias of the repair, and thus determined that the removal of 8-oxodG lesions from mtDNA was not via a transcription-coupled pathway. This was further asserted by measuring the repair in different regions of the mitochondrial genome where the transcription rate differs substantially. The repair in different regions of the mtDNA did not vary, but appeared to be homogeneous w2x. Ligation-mediated PCR has revealed that there are hot spots for oxidative damage to the mitochondrial genome and that the damage removal is not strand biased w19x. Thus, there appears to be agreement that there is no TCR Žof oxidative DNA damage. in mtDNA. A polymerase extension assay has been utilized to evaluate H 2 O 2 damage induction and removal w70x. Both nuclear and mitochondrial sequences were examined. This assay does not measure a specific

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lesion, but rather any damage that blocks the progression of the polymerase. The investigators observed that as the concentration of H 2 O 2 increased, it induced more damage into mitochondrial DNA than nuclear DNA as assessed by the inhibition of the polymerase extension w70x. In contrast, Taffe et al. w61x found no difference in the formation of Fpg-sensitive sites in the mitochondrial and in the nuclear DNA using a different agent, acridine orange. The efficient repair of Fpg-sensitive sites suggested that mitochondria must possess an enzyme capable of recognizing and incising oxidized guanines. In bacterial and mammalian nuclear DNA, oxidative damage at guanines is minimized by the action of 8-oxodG DNA glycosylaserAP lyase enzymes, an adenine DNA glycosylase and an 8oxodGTPase Žfor recent review, see Ref. w13x.. Using purified rat liver mitochondria, we have purified an 8-oxodG DNA glycosylaserAP lyase enzyme, ŽmtODE. that specifically incises 8-oxodG within the context of a 8-oxodG:dC duplexed oligonucleotide w12x. It has the same 8-oxodG:dC substrate preferences as the rat nuclear Ogg1 that was recently purified w43x. However, mtODE does not appear to incise the formamidopyrimidine adduct which is recognized by rat nuclear Ogg1. In addition, when mtODE cleaved an AP site it produced the b elimination product w13x while the rat nuclear Ogg1 appears to generate a b–d elimination product w43x. Also, purified mtODE does not cross react with antibodies generated against the mouse or human forms of Ogg1 ŽCroteau et al., unpublished data.. Therefore, we do not believe that mtODE is the mitochondrially localized form of Ogg1, even though the genes for the mouse and human homologs of Ogg1 have been reported to contain putative mitochondrial targeting signals and shown to be sorted to the mitochondria w47x. Although Ogg1 has been localized to the mitochondria and we have purified an activity that behaves very similarly to Ogg1, we must conclude at this time that there is more than one enzymatic activity operating in mitochondria to facilitate the repair of oxidatively damaged guanines. Incorporation of 8-oxodGTP by DNA polymerases also increases the frequency of this adduct in DNA. 8-oxodG is considered to be a premutagenic lesion because it can mispair with dA during DNA

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replication w31,54,67x. Furthermore, it has been shown that all replicative polymerases readily insert dA opposite 8-oxodG while the DNA repair polymerase b Žpol b . inserts the correct base dC w9,35,39,55x. In vitro extension assays with polg and defined substrates containing an abasic site or 8oxodG revealed that polg Inserts dA opposite either of these substrates at a significant rate w39x. To minimize such mismatches cells possess an adenine DNA glycosylase, hMutY, which excises the incorrect adenine in the context of 8-oxodG:dA mispair. Using an epitope-tagged hMutY, this protein has also been localized to the mitochondria by Takao et al. w62x. In addition to the glycosylases mentioned above, mitochondria also possess an 8-oxodGTPase to minimize the incorporation of the mutagenic nucleotide into newly replicated DNA w26x. Again, it appears that the nuclear gene MTH1 codes for both the nuclear and mitochondrial isoforms of the protein w26x. Although the repair of 8-oxodG has received a considerable amount of attention with respect to mtDNA damage and repair, the repair of other lesions caused by oxidative stress such as pyrimidine hydrates is equally important. Pyrimidine hydrates have been detected in mtDNA w23,73x. One of the pyrimidine hydrates, thymine glycol, can block DNA and RNA polymerases and has been shown to be slightly mutagenic. Repair of thymine glycol from nuclear DNA proceeds through a comparable mechanism as that described for the repair of 8-oxodG. A pyrimidine hydrate DNA glycosylase cleaves the N-glycosylic bond between the sugar and damaged base after which the resulting AP-site is further processed. All known pyrimidine hydrate DNA glycosylases contain an additional AP lyase activity, which incises on the 3X of the AP-site. Pyrimidine hydrate DNA glycosylasesrAP lyases which repair thymine glycols and other pyrimidine lesions have been isolated and cloned from various organisms w3,4,22,24,25,72x, including humans. Recently, we were able to partially purify three enzymatic activities from rat liver mitochondria that specifically cleave thymine glycols in duplex DNA w60x. One of these activities, mitochondrial thymine glycol endonuclease ŽmtTGendo. was further characterized. MtTGendo has an associated AP lyase activ-

ity. The enzyme does not incise 8-oxodG or uracil containing DNA. MtTGendo has a molecular weight around 30 kDa and the most active fractions displayed a single protein band that cross-reacted with a polyclonal antibody raised against E. coli endonuclease III w60x. Recently, the human NTH1 was localized to both the nucleus and mitochondria w62x. Also, it has been proposed that Scr1 ŽNTG1. from S. cereÕisae contains a putative mitochondrial localization signal w4x. Interestingly, the substrate specificity of NTG1 and NTG2 was very similar, although NTG1 also recognizes 8-oxodG:dG w53x. Perhaps repair of this lesion in mitochondria is of critical importance. MtTGendo may be a rat homolog of these mitochondrial forms of pyrimidine hydrate DNA glycosylasesrAP-lyases. Experiments are ongoing to determine whether mtTGendo has affinity for lesions other than thymine glycol. Comparative characteristics of MtODE and MtTGendo are shown in Fig. 2, and the characteristics of the enzymatic cleavage are shown in Fig. 3. 2.3. Repair of alkylation damage Alkylation damage is usually repaired by a BER mechanism. Using the gene specific approach the removal of alkylation damage has been demonstrated in mtDNA. Some of the agents that have been used include: streptozotocin w37x, methylnitrosurea w28x and methyl methanesulfonate w40x. Except for the NER deficient cell lines, for each of these agents the repair in the mtDNA was equivalent to that observed in the nuclear genome. Nitrogen mustard ŽbisŽ2-chloroethylmethyl.amine. also causes alkylation damage, however repair of this more complex alkylation damage is not repaired in mtDNA w28x. In nuclear DNA, complex alkylation damage is thought to be repaired by the NER machinery and as will be discussed below, NER as we know it in the nucleus, does not appear to operate in mitochondria. 2.4. Repair of abasic sites Abasic sites are formed because of various chemicals, by DNA glycosylases ŽUDG for example., and by spontaneous hydrolysis of the bases. AP endonucleases are classified according to where they cleave the phosphodiester bond relative to the abasic site w15x. Class I enzymes cleave on the 3X side of the

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Fig. 2. Oxidative damage specific mitochondrial AP lyases from rat liver.

abasic site leaving an unsaturated baseless sugar and a 5X phosphate. Class II enzymes cleave 5X to the abasic site leaving 3X OH and 5X-deoxyribose phosphate Ž5X dRp. ends. Two mitochondrial class II AP endonucleases have been purified from mouse plasmacytoma cells w64x. Both activities cross-reacted

with antibodies raised against the major AP endonuclease of HeLa cells and corresponded to bands that were 66 kDa in size, while the cytoplasmic form of the major AP endonuclease from mouse is 41 kDa. As discussed by the authors, it is unlikely that the mitochondrial AP endonucleases were derived from

Fig. 3. Cutting with mtODE and mtGendo.

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the nuclear gene for the major AP endonuclease. Further support for that conclusion came from Takao et al. w62x who failed to see mitochondrial localization of hAPE. Clarification awaits the cloning of the mitochondrial AP endonuclease genes. Recently, Pinz and Bogenhagen w38x have reconstituted the repair of abasic sites in vitro using purified mitochondrial components. They used a mitochondrial class II AP endonuclease, a deoxyribophosphodiesterase, mtDNA polymeraseg Žpolg . and a DNA ligase each purified from Xenopus laeÕis mitochondria. The AP endonuclease was similar to the mouse AP endonuclease described above. Polg was able to produce a 1-nucleotide replacement following the AP endonuclease activity. Their assay system also revealed that both DNA polg and the mtDNA ligase possess the ability to remove a 5X-32 PdRp residue. Another study by Longley et al. confirms that human DNA polg possesses AP lyase activity w29x. Pinz and Bogenhagen have also isolated the 100 kDa mtDNA ligase to homogeneity w38x. They suggest that the mtDNA ligase may be generated from an alternative product of the DNA ligase III gene w38x. 2.5. Damage reÕersal The most direct form of DNA repair is the chemical reversal of the aberrant DNA adducts ŽTable 1.. Photolyases use light to monomerize UV-induced cyclobutane pyrimidine dimers in DNA. Yeast photolyase localizes to both the nucleus and mitochondria w71x. However, in higher eukaryotes, it is un-

Table 1 Type of DNA repair mechanism

Nucleus

Present in: Mitochondria

Photolyase DNA methyl transferase Base excision repair Alkylation damage Oxidative damage Single strand break Mismatch repair Nucleotide excision repair Transcription-coupled repair Recombinational repair

v v

v v

v v v v v v

v v v ? no no

v

?

known if photolyases exist in mitochondria. Mitochondrial lysates derived from X. laeÕis oocytes may possess photolyase activity. Ryoji et al. w49x employed a PCR based in vitro assay to determine whether mitochondrial extracts could repair UVirradiated DNA. They observed that if the mitochondrial lysate and UV-irradiated DNA were incubated under light, then the substrate DNA became a better PCR template, which suggests a photoreactivation mechanism for UV-damage. The mitochondrial protein responsible for this photoreactivation will most likely be isolated soon. Another form of direct reversal is mediated by DNA methyltransferases, also named DNA alkyltransferases. These ‘suicide’ enzymes transfer the chemical adduct directly to a cysteine residue within the enzyme and thereby inactivate themselves. Mammalian alkyltransferases have been shown in vitro and in vivo to remove methyl and larger alkyl adducts such as ethyl groups from DNA w36x. An O 6-methylguanine-DNA methyltransferase has been partially purified from rat liver mitochondria w32x. In this study the authors investigated the formation and removal of O 6-methyl-2X-deoxyguanine and O 6butyl-2X-deoxyguanine from nuclear and mtDNA. The kinetics of removal for the O 6 -methyl-2X-deoxyguanine were similar between the mtDNA and nuclear DNA while there was no removal of the bulkier O 6-butyl-2X-deoxyguanine. Further support for mitochondrial methyltransferases comes from work by Satoh et al. w50x. They reported the removal of O 6-ethyl-2X-deoxyguanosine from rat liver mtDNA following exposure to N-ethyl-N-nitrosourea in vivo. It is unknown what the relative contribution is of base excision repair and alkyltransferases to the repair of smaller alkylation adducts within mtDNA. 2.6. Mismatch repair Mismatch repair in bacteria is mediated by mut L, mut H and mut S. Homologs of these proteins have been identified in yeast and humans. While mismatch repair has not been directly measured in mitochondria from higher eukaryotes, in yeast it clearly does exist. In S. cereÕisae a mut S homolog, MSH1, was identified because it caused gross mtDNA rearrangements and an increased rate of mtDNA mutagenesis w45x. Given the phenotype of the yeast with a

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defective Mut S homolog w45x, it is unlikely that such a mutation in humans would be compatible with life. Further investigation of mismatch repair in mitochondria needs to be explored. 2.7. Recombinational repair Whether or not a recombination repair mechanism exists in mitochondria is controversial. Interstrand cross-links are thought to be repaired by recombinational repair, and some types of these lesions are repaired in mitochondria while others are not. As discussed above, the cisplatin interstrand cross-links are repaired w28x while psoralen interstrand crosslinks are not w14x. By the identification of recombination intermediates, homologous recombination has been postulated to occur between mitochondrial genomes in Kearns–Sayre syndrome and chronic external ophthalomoplegia patients w42x. However, cell fusion experiments have demonstrated that recombination between mtDNA molecules does not generally occur ŽRef. w42x and references sited therein.. Also, an in vitro study suggests that mitochondria do possess a homologous recombination pathway w63x. Thus, recombinational repair in mitochondria remains controversial and its definitive existence awaits further research. Given that an in vitro assay has already been developed, identification of the proteins involved can be undertaken. 2.8. Nucleotide excision repair NER is responsible for removing a large number of adducts from DNA Žfor a recent review, see Ref. w69x.. There are numerous polypeptides involved in the NER pathway. When analyzing the NER pathway, the adduct of choice has often been UV-induced DNA damage. In 1974, Clayton et al. w10x reported that UV-induced pyrimidine dimers were not removed from mtDNA. This study has been confirmed in a variety of cell types and was the first to suggest that mitochondria lacked NER machinery. Consequently, bulky adducts are not generally repaired from the mitochondrial genome. Cisplatin is a commonly used anticancer drug that is repaired by the NER pathway in nuclear DNA. It generates both intrastrand Žthe major lesion. and interstrand Žminor lesion. DNA cross-links. The re-

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pair of cisplatin intrastrand cross-links, as measured by a DNA relaxation assay or the gene specific repair assay, revealed that the major adduct, the intrastrand adduct is not repaired in mtDNA w28,56x. However, the interstrand cisplatin adduct appears to be repaired in mtDNA w28x. The interstand cross-link repair may be repaired via a recombination repair mechanism although NER is often considered to be involved. The carcinogen 4NQO is known to be repaired by NER in mammalian cells. Although it is a bulky carcinogen, it invokes the oxidative stress response in E. coli. In contrast to the other bulky lesions, this one is removed efficiently from the mtDNA w59x. Evidence suggests that more than one mechanism exist for the repair of 4-NQO damage. In bacteria, the Fpg protein provides protection against 4-NQO damage and the authors showed that 8-oxodG was produced by 4-NQO treatment w48x. This provocative finding of 4NQO repair in mtDNA requires further study to clarify whether any NER proteins may be involved. 2.9. Degradation or repair The question as to whether mitochondria actually repair damaged genomes or degrade them is still open for debate and depends in part on the damage being evaluated. Mitochondria contain multiple genomes, and perhaps it is not necessary to repair the damage within all copies since they can functionally complement each other. Damaged genomes can either be repaired or degraded as a means of preserving the DNA’s coding integrity. Although degradation may be the best method for some lesions, clearly with the constant exposure to oxidative damage such damage must, to a large extent, be repaired. Recent studies in this laboratory suggest that there is no significant mitochondrial degradation during the repair of singlet oxygen DNA damage w2x, at least in some mammalian cell lines. A similar observation was made in the LMPCR analysis of alloxan DNA damage and repair study w19x. In vivo the normal exposure of mtDNA to agents which cause UV or bulky DNA lesions may not be significant and therefore degradation may be the best choice for such rare lesions. However, the consequences for this coping strategy may not be so

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advantageous when normal exposure levels are increased. Some of the adverse effects of cisplatin chemotherapy have been attributed to mitochondrial dysfunction w57x. 2.10. Repair of mitochondrial DNA in Õitro Some of the studies mentioned above were done in cell culture and thus resembles the in vivo situation more. Much of the recent progress in the mechanistic studies on DNA repair has been done utilizing in vitro approaches. Such experiments are done using purified proteins in simple reactions involving a very limited number of components. The function of mitochondrial repair enzyme are being tested in a reconstituted base excision repair pathway based on knowledge about the nuclear processes w29,38x. Mitochondrial base excision repair has been reconstituted with purified enzymes, mitochondrial uracil DNA glycosylase, AP endonuclease, DNA polymerase and ligase. This approach is bound to provide us with much molecular insight in the future about the precise mode of action of some of the enzymes involved. The use of reconstituted systems, in which a preconceived mechanistic notion is tested, has its limitations. It can be argued that these systems do not reflect the in vivo repair process. We are now developing alternative approaches using whole mitochondrial protein extract to study the repair of defined DNA lesions. In the study of mammalian nuclear DNA repair, this approach has proven extremely useful. Damaged plasmid is added to a cell extract, and the repair incorporation into the plasmid is then measured after the repair incubation period. With this approach, the repair of a wide variety of lesions can be assessed, and the measurements resemble the repair situation better as it occurs in vivo w68x. While these experiments have been carried out for nuclear repair over the last decade, it has still not been done in mitochondria extracts. In one report w49x, using Xenopus extracts, this was attempted, but not optimized, and no specific lesions were studied. It has been a challenge to get this experiment to work with mammalian mitochondrial extracts, presumably because of the presence of a potent endonuclease, most likely endonuclease G, that readily digests DNA containing strings of guanine.

We have recently been able to measure repair in vitro in mitochondrial extracts, and we are now getting stable repair incorporation into plasmids containing single uracil lesions, which are known to be substrates for base excision repair ŽStierum et al., in prep.. This approach lends itself to many important experiments where different DNA lesions are assayed and where biochemical mechanisms of mitochondrial repair are explored.

3. Aging Several investigators have reported the accumulation of 8-oxodG in DNA with age but there are conflicting reports as to the degree of accumulation. One of the controversies in the study of oxidative DNA damage concerns the amount of 8-oxodG present in mtDNA. Although there appears to be a consensus about an increase of damage with age, the amounts of oxidative base modifications measured by various methods ŽLCrMS, HPLC, and enzymatic. do not agree with one another w11x. There is a need to make concerted efforts to measure oxidative lesions and their repair under identical conditions and in the same biological system using different methods to assess the same changes. The mitochondrial theory of aging postulates that organisms age due to the accumulation of DNA damage and mutations in the multiple mitochondrial genomes, leading to mitochondrial dysfunction. Among the many types of DNA damage, 8-oxodG has received the most attention due to its mutagenicity and because of the possible correlation between its accumulation and pathological processes like cancer, degenerative diseases and aging. Although 8oxodG accumulation with age in the mtDNA has been well documented, very little is known about its processing and no published study has yet examined whether mitochondrial DNA repair changes with age. We have assessed the age related changes in the capacity for mtDNA repair by measuring endonucleolytic activity towards an 8-oxodG containing substrate. This activity is, as discussed above mediated by mtODE. Extracts from purified rat heart and liver mitochondria were used to measure the activity of this enzyme that specifically cleaves 8-oxodG-containing duplex oligonucleotides. We find that this

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activity is higher in 12 and 23 months old than in 6 months old rats, in both liver and heart extracts w74x. Our results suggest that the mitochondrial capacity to repair 8-oxodG, the main oxidative base damage shown to accumulate with age in mtDNA, does not decrease, but rather increases with age. We also tested the activity of other repair enzymes, such as uracil DNA glycosylase, in mitochondrial extracts from rats of different ages, and found no age related changes. The specific increase in 8-oxodG endonuclease activity, rather than a general up-regulation of DNA repair in mitochondria, suggests the possibility of an induction of this repair pathway with age w74x.

4. Mitochondrial repair defects in human premature aging disease In the molecular study of the aging process, a category of human diseases has been particularly useful. These are the so-called premature aging disorders where the patients appear much older than they actually are. Some of these conditions appear to be natural human mutants, and the mutated gene has been identified. This is the situation for xeroderma pigmentosum ŽXP., Cockayne syndrome, Werner syndrome and others. If the aging process has progressed in these disorders, it is of particular interest to study mtDNA repair based on the above considerations. The defective repair of oxidative damage in mtDNA has been reported for XP-A cells Ža NER deficient strain. w18x. The XP-A protein participates in the damage recognition phase of the NER pathway and cell lines defective in the XP-A protein are the most damage sensitive NER deficient strains. Driggers et al. w18x used alloxan to generate oxidative damage on wild type and XP-A cells grown in culture. They then used the gene specific repair assay to evaluate the rate of repair within the mitochondrial genome and the nuclear gene DHFR. The repair of alkali and enzyme-sensitive sites in mtDNA from XP-A cells was approximately 40–50% that seen in the mtDNA from WI-38 cells. Such findings are inconsistent with the findings that NER does not exist in mitochondria. A more comprehensive study of the XP cell lines should be undertaken because there are variations within the various XP lines and at this time we do not know if the above observation

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is unique to that cell line or is more applicable to all XP-A cell lines. Down’s syndrome ŽDS. or trisomy 21 patients are characterized by premature aging. Increased mortality is observed after the age of 40 as compared to normal individuals. Using the gene-specific DNA repair technique, Druzhyna et al. w21x studied the repair of oxidative stress-induced mtDNA damage in cells obtained from DS patients and normal individuals. They observed that menadione-induced alkalisensitive sites, which include AP-sites that may arise from depurinated oxidized bases, were less efficiently removed from mtDNA in DS than in normal cells. With the caveat that only a very low initial amount of DNA damage was induced Ž0.7–0.8 breaksr13.5 kb fragment. which may complicate accurate assessment of the repair, this study suggests that decreased mtDNA repair contributes to the observed clinical features in DS.

5. Conclusions Mammalian mitochondria clearly possess a number of DNA repair mechanisms. However, the repertoire of adducts that can be removed from the mitochondrial genome is clearly not as diverse as that of the nucleus because mitochondria appear to lack NER as we know it from the nuclear repair. In the near future, we hope more work will concentrate on the identification and cloning of other specific DNA repair enzymes. Thus, we will be better able to predict what kinds of damage will be repaired within mitochondria. The mitochondrial theory of aging suggests that it is the mitochondria within our cells which determines how long cell live w30x. The accumulation of damage to the mitochondrial genome plays a central role in this theory, yet we know little about whether DNA repair activities increase or decrease with age. Consequently, this laboratory has begun to evaluate how the activities of the individual mitochondrial repair proteins that we have isolated change with respect to age. The field of mitochondrial DNA repair is just awakening and it will be exciting to investigate what types of DNA repair proteins exist in mitochondria, how they are regulated and how they get there.

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Acknowledgements We appreciate the interaction with the Danish Center for Molecular Gerontology.

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