Detection of DNA Base-Excision RepairActivity for Oxidative Lesions in Adult RatBrain Mitochondria

Dexi Chen,1,2 Jing Lan,1,2 Wei Pei,1,2 and Jun Chen1–3*1Department of Neurology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania2Institute for Brain and Aging, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania3Geriatric Research, Educational and Clinical Center, Veterans Affairs Pittsburgh Health Care System,Pittsburgh, Pennsylvania

Endogenous oxidative damage to brain mitochondrialDNA and consequential disturbances of gene expressionand mitochondrial dysfunction have long been impli-cated in aging and the pathogenesis of neurodegenera-tive diseases. It has yet to be determined, however,whether mitochondria in brain cells contain an activeDNA repair system and, if so, how this system functions.Therefore, the capacity for the repair of defined types ofoxidative DNA lesions has been investigated in adult ratbrain mitochondria. Using in vitro DNA incorporationrepair assay, we have detected base excision repair(BER) activity for the common oxidative DNA adduct8-hydroxyl-29-deoxyguanine (8-oxodG) in mitochondriaprotein extracts from cortical tissues and cultured pri-mary cortical neurons and astrocytes. The levels of BERactivity were both protein concentration-dependent andrepair-incubation time-dependent. To resolve the BERpathway, the activity of essential BER enzymes was ex-amined in mitochondria using oligonucleotide incisionassay, DNA polymerase assay, and DNA ligase assayemploying specific DNA substrates. Mitochondrial ex-tracts were able to remove specifically 8-oxodG, uracil,and the apurinic/apyrimidinic abasic site from sub-strates. Moreover, a gamma-like DNA polymerase activ-ity and a DNA ligase activity were detected in mito-chondiral extracts, based on the formation of specificrepair products. These results demonstrate that adultbrain mitochondria possess an active BER system forrepairing oxidative DNA lesions. This repair system ap-pears to function by sequential actions of DNA repairenzymes that are homologous to, but not identical to,that in the nucleus. Thus, BER may represent an endog-enous protective mechanism against oxidative damageto mitochondrial, as well as nuclear, genomes in braincells. J. Neurosci. Res. 61:225–236, 2000.© 2000 Wiley-Liss, Inc.

Key words: mitochondria; oxidative stress; DNA dam-age; base excision repair; brain

Mitochondrial DNA (mtDNA) encodes 13 func-tional proteins and is the only genome found outside the

nucleus. All of the mtDNA-encoded proteins are directlyinvolved in oxidative phosphorylation and are crucial tothe normal function of the respiratory chain. mtDNA isparticularly susceptible to and constantly damaged by cel-lular reactive oxygen species (ROS) for several reasons.First, mitochondria consume about 90% of oxygen in thecell, and about 2% of the oxygen metabolized in themitochondria is converted to superoxide and hydrogenperoxide (Richter, 1992); thus, mitochondria are the larg-est subcellular source for the production of ROS. Second,mtDNA is located in close proximity to the inner mito-chondrial membrane, where ROS are generated as by-products of the electron transport chain. Third, unlike thenuclear DNA, mtDNA lacks protection from histones.Oxidative damage to mtDNA and its consequences, suchas gene mutations and deletions, have long been impli-cated in the pathogenesis of a variety of human disordersthat are associated with mitochondrial dysfunction andwith aging (Tritschler and Medori, 1993). Thus, effectiveDNA repair function is essential for the cell to maintainthe integrity of mitochondrial genome and, thus, thenormal mitochondrial energy metabolism.

Several studies have suggested that, as with the nu-cleus, mammalian mitochondria have the capacity to re-pair oxidative DNA damage to their genome (Croteau andBohr, 1997). Several independent groups have reportedthat mammalian mitochondria are efficient at repairingcertain oxidative DNA damage, such as base damage,single-strand breaks, and apurinic/apyrimidinic sites (Taffeet al., 1996; Driggers et al., 1997). The DNA base excisionrepair (BER) pathway appears to be the predominant, ifnot the exclusive, mechanism for the repair of oxidativeDNA lesions in the mitochondria (Croteau and Bohr,1997). Evidence supporting BER capacity in mitochon-dria also has been derived from the fact that some enzyme

*Correspondence to: Dr. Jun Chen, Department of Neurology, S506-Biomedical Science Tower, University of Pittsburgh School of Medicine,Pittsburgh, PA 15213.E-mail: jun@med.pitt.edu

Received 16 February 2000; Revised 7 April 2000; Accepted 12 April 2000

Journal of Neuroscience Research 61:225–236 (2000)

© 2000 Wiley-Liss, Inc.

activities necessary for BER have been identified in mi-tochondria (Croteau et al., 1997; Tomkinson et al., 1988;Pinz and Bogenhagen, 1998).

Thus far, characterization of the mammalian mito-chondrial BER mechanism has been limited to nonneu-ronal tissues and cell lines. A number of studies havesuggested that the brain contains higher steady-state levelsof several, but not all, forms of endogenous oxidativemtDNA lesions compared to other organs (Ames et al.,1993). The accumulation of oxidative DNA lesions inbrain mitochondria, which occurs mainly in regions hav-ing a higher metabolic rate, shows marked age dependence(Corral-Debrinski et al., 1992; Cortopassi et al., 1992;Zhang et al., 1992; Mecocci et al., 1993). UnrepairedmtDNA damage can affect normal mitochondrial geneexpression and thus may contribute to an age-dependentdecrease in mitochondrial oxidative phosphorylation(Harmon et al., 1987; Corral-Debrinski et al., 1992;Bowling et al., 1993). These changes in brain mitochon-dria have been thought to participate in the pathogenesisof various neurodegenrative disorders (Parker et al., 1990;Shoffner et al., 1991; Corral-Debrinski et al., 1994; Swer-dlow et al., 1996). Despite the apparent importance ofoxidative damage to mtDNA in the development of mi-tochondrial dysfunction involved in neurodegeneration,however, virtually no information is currently availableregarding whether or how the DNA repair system func-tions in brain mitochondria. As a first effort to explorethe mechanism of the brain mtDNA repair process and,eventually, to elucidate how pathological alterations ofmtDNA repair function might contribute to neurologicaldisorders, we have studied the BER pathway in adult ratbrain mitochondria. DNA containing various base lesions,including alkylating lesions, uracil, and oxidative lesions,has previously been used as specific substrates in various invitro repair assays for the measurement of cellular BERactivity (Satoh and Lindahl, 1994; Sobol et al., 1996). Inthe present study, the pcDNA plasmid carrying the com-mon oxidative base lesion 8-hydroxyl-29-deoxyguanine(8-oxodG) was used as the substrate for examining theoverall BER activity in mitochondrial protein extracts.Various DNA repair assays were then employed to detectthe activities of individual repair enzymes involved in theBER pathway.

MATERIALS AND METHODS

Isolation of Mitochondria and Mitochondrial Protein

Mitochondria were isolated from brains of adult maleSprague-Dawley rats weighing 275–300 g (Hilltop Sprague-Dawley, Scottdale, PA). All experimental procedures were car-ried out under protocols approved by the Animal Care Com-mittee of the University of Pittsburgh and in accordance withthe principles outlined in the National Institutes of HealthGuide for the Care and Use of Laboratory Animals.

All procedures for mitochondria and mitochondrial pro-tein isolation were carried out at 4°C. The isolation method wasadapted from methods previously described (Sun and Gilboe,1994; Croteau et al., 1997). In brief, brain cortical tissues taken

from the frontal-parietal cortex, approximately 1 g per sample,were minced and homogenized using a Dounce homogenizer in13 M-SHE buffer containing 0.21 M manitol, 0.07 M sucrose,10 mM Hepes-KOH at pH 7.4, 1 mM EDTA, 0.15 mMspermine, and 0.75 mM spermidine. The following freshlyprepared protease inhibitors were then added: 1 mg/ml each ofleupeptin, aprotinin, and pepstatin; 1 mM PMSF; and 1 mMDTT. After lysis for 30 min on ice, unbroken cells and nucleiwere pelleted at 1,200g. The supernatant, containing the mito-chondria, was centrifuged at 10,000g for 15 min to pellet themitochondria. To isolate mitochondrial protein further, thepellet was resuspended in a solution containing 3% Ficoll 400,0.12 M mannitol, 0.03 M sucrose, and 25 mM EDTA (pH 7.4)and gently layered twice in 6% Ficoll 400 solution to produce adiscontinuous density gradient. After centrifugation at 10,400gfor 25 min, the sediment was resuspended in 13 M-SHE buffercontaining 3 mg/ml of digitonin for 15 min. This was followedby centrifugation at 10,500g for 15 min. The pellet was washedwith 13 M-SHE buffer and then lysed for 30 min in a lysisbuffer containing 20 mM Hepes (pH 7.4), 400 mM KCl, 1 mMEDTA, 5% glycerol, 0.5% Triton X-100, 2 mM DTT, 0.5 mMPMSF, and 1 mg/ml each of leupeptin, aprotinin, and pepstatin.The lysate was centrifuged at 130,000g for 1 hr, concentrated to5–10 mg/ml protein in a speed vacuum vaporizer (modelSC100; Savant Inc., Farmingdale, NY) at 4°C, and stored at280°C.

Determination of the Purity of MitochondrialProtein Preparation

To determine the purity of the Ficoll-400 gradient-purified mitochondrial protein, two types of assays were per-formed to determine the levels at which mitochondrial proteinpreparation was contaminated with cytosolic or nuclear pro-teins. For the detection of cytosolic proteins, lactate dehydro-genase (LDH) activity was assayed spectrophotometrically inmitochondrial protein and crude whole-cell protein extractsusing the 340-LD kit (Sigma, St. Louis, MO) according to themanufacturer’s instructions. Western blot analysis was per-formed using a monoclonal antibody against the cytosolic pro-tein b-actin (Sigma). For the detection of nuclear proteins,Western blots were performed to analyze poly(ADP-ribose)-polymerase (PARP) and proliferating cell nuclear antigen(PCNA). The monoclonal antibodies against PARP (Biomol,Plymouth Meeting, PA) and PCNA (PharMingen, San Diego,CA) were used at working dilutions of 1:5,000 and 1:1,000,respectively, as suggested by the manufacturers. Western blotswere also performed to detect the mitochondrial markers usingmonoclonal antibodies against cytochrome c (PharMingen) at aworking dilution of 1:5,000 and cytochrome c oxidase subunitIV (Molecular Probes, Inc., Eugene, OR) at a dilution of1:4,000. For the assessment of nonspecific immunostaining, aduplicate blot from each experiment was incubated without theprimary antibody.

In Vitro DNA Incorporation Repair Assay

The method originally described by Wood (1989), usingcell protein extracts to repair defined types of damage in plasmidDNA, has been used extensively to study DNA excision repairactivity in various cell systems. We have applied this method to

226 Chen et al.

study the ability of mitochondrial protein extracts to incorporate[32P]dGTP into oxidatively damaged plasmids. The DNA repairsubstrate used in the present study was a pcDNA plasmid (Strat-agene, La Jolla, CA) containing the oxidative adduct 8-oxodG,a common DNA lesion known to be repaired by the DNA baseexcision repair pathway. These experiments provided an esti-mation of overall BER activity in the mitochondria.

Preparation of DNA repair substrates. pcDNAplasmids were transfected into E. Coli DH5a cells (Gibco BRL,Gaithersburg, MD). Transfected cells were grown at 37°C for12 hr and collected by centrifugation at 5,000g for 10 min. Theplasmid DNA was purified from the transformed cells using alarge-scale DNA purification kit (Promega, Madison, WI) ac-cording to the manufacturer’s instructions. The closed circularfraction of pcDNA plasmids was isolated via CsCl gradientcentrifugation using a Beckman LE-80 ultracentrifuge equippedwith a Ti70.1 rotor (Sambrook et al., 1989). Ethidium bromidewas subsequently removed from the DNA by five cycles ofextraction with 1-butanol saturated with water. CsCl was re-moved from the DNA solution by diluting with 3 volumes ofwater and precipitating the DNA with 2 volumes of ethanol for30 min at 4°C, followed by centrifugation at 10,000g for 15 minat 4°C. The precipitated DNA was resuspended in the TE buffer(pH 8.0), subjected to OD260 measurement, and then stored at220°C. For the induction of 8-oxodG in the plasmid DNA, aprocedure using photoactivated methylene blue (MB) as thelesion inducer was carried out as previously described (McBrideet al., 1992). The purified pcDNA plasmids were diluted in10 mM Tris-HCl and 1 mM EDTA (pH 8.0) to a final con-centration of 100 mg/ml. One-ninth volume of 20 mM MB wasadded into the DNA solution in the dark and mixed rapidly, andthe mixture was placed in a 10-cm plastic tissue culture dish onice. The uncovered dish was exposed to light from a 100 Wtungsten light bulb positioned 20 cm from the surface for 30min. The DNA was ethanol-precipitated three times to removeMB and then resuspended in TE buffer. All procedures werecarried out under a dim blue light while MB was present insolution with the DNA. The content of 8-oxodG in plasmidswas then measured using high-performance liquid chromatog-raphy with electrochemical detection (HPLC-EC) as previouslydescribed (Lan et al., 2000).

In vitro DNA repair assay. Mitochondrial extracts(amounts of protein as indicated in the experimental protocol)were incubated for a period of time as indicated in the protocolat 30°C in 50 ml of reaction mixtures containing 0.3 mg each ofdamaged pcDNA plasmids containing the 8-oxodG lesions,45 mM HEPES-KOH (pH 7.8), 70 mM KCl, 5 mM MgCl2,1 mM DTT, 0.4 mM EDTA, 2 mM ATP, 20 mM each ofdATP, dTTP, and dCTP, 8 mM dGTP, 2 mCi of [a-32P]GTP(NEN, Boston, MA), 40 mM phosphocreatine, 2.5 mg creatinephosphokinase, 3% glycerol, 20 mg/ml BSA, 2 mM NAD1, and1 mM b-mercaptoethanol. In some experiments, undamagedpSPORT1 plasmids served as negative controls. The reactionwas terminated by the addition of proteinase K (240 mg/ml),SDS (to 1%), and EDTA (to 20 mM) and incubation of thesamples for 30 min at 37°C. Plasmid DNA was phenol-extractedfrom the mixture, 5 mg of carrier tRNA was added and theDNA precipitate was dissolved in 20 ml of TE buffer. The

samples were treated with 10 units of BamHI (Gibco BRL,Grand Island, NY) overnight at 37°C to linearize the DNA andthen separated by electrophoresis on a 1% agarose gel. DNAbands on the gel were visualized using UV light and photo-graphed. Radioactive nucleotide incorporation into the DNAwas detected using autoradiography. Autoradiogram signals onthe films were semiquantified (optical density 3 area) by a geldensitometric scanning program using the Microcomputer Im-aging Device (MCID) image analysis system (St. Catharine’s,Ontario, Canada). All densitometric values for DNA radiolabelswere normalized to values for UV photographs of DNA bandson the same lane.

In Vitro Oligonucleotide Incision Assay

The following experiments, including the in vitrooligonucleotide incision assay, DNA polymerase-g assay,and DNA ligase assay were designed to detect the repairactivities of mitochondrial protein extracts along the BERpathway. The in vitro oligonucleotide incision assay wasuse to estimate the ability of mitochondrial protein extractsto recognize and remove defined types of oxidative DNAlesions. The incision of two types of DNA lesions wastested in the present study: 8-oxodG and apurinic/apyrimidinic (AP) abasic site. The DNA substrate used inthis assay was a 21-mer oligodeoxynucleotide containingeither an 8-oxodG or AP site at position 11 (for sequencessee Figs. 4, 5). The oligonucleotide containing either theoxidative lesion or normal unmodified bases was 59-end-labeled using T4 polynucleotide kinase and [g-32P]ATP,and the reaction mixture was passed through a G-25 spincolumn (5 prime 3 3 prime, Inc., Boulder, CO) toremove the free unlabeled [g-32P]ATP. The labeled oli-gonucleotide was then annealed to the complementaryoligonucleotide in 100 mM KCl, 10 mM Tris (pH 7.8),and 1 mM EDTA by heating the oligonucleotides to 80°Cand then allowed to cool slowly to room temperature. Inthe case of the oligonucleotide that contained an AP site,the sample was heated to 55°C instead of 80°C, because ofthe heat lability of the AP site. The reaction mixture forthe incision assay contained 40 mM Hepes-KOH (pH7.6), 75 mM KCl, 2 mM DTT, 1 mM EDTA, 0.1 mg/mlBSA, 5 nM 32P-labeled DNA duplex, and mitochondrialextracts or purified enzymes at the amount indicated ineach experiment. The reaction was carried out at 37°C fora period of time according to the experimental protocol,then terminated by the addition of an equal volume ofloading buffer containing 90% formamide, 0.002% brom-phenol blue, and 0.002% xylene cyanol. The sample washeated to 80°C for 2 min and subjected to electrophoresison a denaturing 20% polyacrylamide gel containing 7 Murea. After electrophoresis, the gel was subjected to auto-radiography and densitometry analysis.

DNA Polymerization Assays

DNA polymerase-g assay. DNA polymerase-g ac-tivity in mitochondrial protein extracts was assayed as previouslydescribed (Insdorf and Bogenhagen, 1989). The 23-mer M13/PUC forward sequence primer was annealed to the single-stranded M13 ssDNA (Gibcol BRL) in 100 mM KCl, 10 mM

DNA Repair in the Brain Mitochondria 227

Tris (pH 7.8), and 1 mM EDTA using the heating and slowcooling procedure. The double-stranded DNA was then incu-bated in the reaction mixture at 32°C for 30 min. The reactionmixture contained 45 mM HEPES-KOH (pH 7.8); 40 mMNaCl; 5 mM MgCl2; 1 mM DTT; 2 mM ATP; 1 mM EDTA;40 mM phosphocreatine; 2.5 mg creatine phosphokinase; 20 mMeach of dATP, dTTP, and dGTP; 20 mCi of a-[32P]dCTP; 3%glycerol; 200 mg/ml BSA; 1 mM b-mercaptoethanol; and mi-tochondrial extracts (the amount of protein as indicated in theexperimental protocol). The reaction was terminated by adding5 mg of proteinase K and 0.8 ml of 10% SDS, and the mixturewas incubated for 15 min at 55°C. The reaction product wasthen purified using a G50 column and subjected to scintillationcounting. One unit of activity was defined as the amount ofenzyme that catalyzes the incorporation of 1 pmol of CMP intothe double-stranded DNA.

Primer extension assay. The oligonucleotides usedin these studies (sequences shown in Fig. 6c) were custom-madeand PAGE purified (Biosynthesis, Dallas, TX). The 26-merM26AS oligodeoxynucleotide was 59-end-labeled using T4polynucleotide kinase and [g-32P]ATP (specific activity 5 3 106

cpm/pmol) and purified using a G-25 spin column. The labeledoligomer was then annealed to the 50-mer M50S oligonucleo-tide as described above. The primer extension assay was per-formed as previously described (Torri et al., 1994). The reactionmixture consisted of 45 mM HEPES-KOH (pH 7.8); 40 mMNaCl; 5 mM MgCl2; 1 mM DTT; 2 mM ATP; 1 mM EDTA;40 mM phosphocreatine; 2.5 mg creatine phosphokinase; 20 mMeach of dTTP, dCTP, and dGTP; 3% glycerol; 20 mg/ml BSA;and 1 mM b-mercaptoethanol. Mitochondrial extracts wereincubated with the annealed DNA in the reaction mixture for30 min at 32°C. The reaction was terminated by adding 10 mlof loading buffer containing 90% (vol/vol) formamide, 0.1%(wt/vol) bromophenol blue, and 20 mM EDTA and thenheating at 80°C for 10 min. The reaction products were sepa-rated by electrophoresis on a 15% polyacrylamide gel containing7 M urea and detected using autoradiography.

DNA Ligation Assays

DNA ligase activity in the mitochondrial protein extractswas detected by monitoring the mechanism-based adenylationof enzyme as previously described (Pinz and Bogenhagen,1998). Mitochondrial extracts at amounts indicated in eachexperiment were incubated in a reaction mixture containing20 mM Tris (pH 8.0), 40 mM NaCl, 5 mM MgCl2, 5 mMDTT, 8% glycerol, 0.02% Triton X-100, and 2 mCi of[a-32P]ATP for 15 min at 30°C. The reaction products wereisolated by passage through a Sephadex G-25 column, whichwas equilibrated with a buffer containing 10 mM HEPES (pH7.4), 1.5 mM MgCl2, 10 mM KCl, and 1 mM EDTA and thenaddition of an equal volume of SDS sample loading buffer.Proteins were resolved by electrophoresis on a 10% polyacryl-amide gel containing SDS and detected using autoradiography.

DNA ligase activity in mitochondrial protein extracts wasalso examined using the oligonucleotide ligation assay as previ-ously described (Pinz and Bogenhagen, 1998). This was done byincubation of mitochondrial extracts with DNA substrates pre-pared by annealing 10 pmol of 59-32P-oligo(dT)18 to 1 mg ofpoly(rA)100 in reaction mixtures containing DNA ligase buffer

and 1 mM ATP. The ligase buffer consisted of 20 mM Tris (pH8.0), 40 mM NaCl, 5 mM MgCl2, 5 mM DTT, 8% glycerol,and 0.02% Triton X-100. Ligation products were analyzed byelectrophoresis on a 10% polyacrylamide gel containing 8 Murea and detected by autoradiography.

Data Analysis

All quantitative data are reported as mean 6 SEM. Com-parisons of DNA BER activity, oligonucleotide incision activ-ity, and DNA polymerase activity among different experimentalconditions were made using ANOVA and post hoc Bonferroni/Dunn tests. A level of P , 0.05 was considered statisticallysignificant.

RESULTS

Purity of the Mitochondrial Protein PreparationWe have measured the concentration of lactate de-

hydrogenase (LDH) in the crude whole-cell homogenateand in the Ficoll-400 gradient-purified mitochondrial pro-teins. The ratio of LDH concentration between crudewhole-cell homogenate and mitochondrial extracts wasgreater than 30:1 in all tested samples prepared either fromcerebral cortical tissues or from primary neuronal cultures,being comparable to that of rat liver mitochondrial prep-arations reported by others (Croteau et al., 1997).

To determine further whether the mitochondrialextracts were contaminated with detectable amounts ofnuclear or other cytosolic proteins, a panel of Westernblots was performed to detect b-actin, PARP, PCNA, andtwo mitochondrial markers, cytochrome c and cyto-chrome c oxidase. Immunoreactivity of cytochrome c andcytochrome c oxidase but not any of the three cytosolic ornuclear proteins was readily detectable in the mitochon-drial protein preparations (Fig. 1).

In Vitro DNA Base Excision Repair Activity inBrain Mitochondrial Extracts

Photoactivated MB was used to induce oxidativedamage to the plasmid DNA, 8-oxodG being the predom-inant product of this reaction (Schneider et al., 1990). Inthis study, 30 min of exposure to photoactivated MBresulted in an average 55-fold increase in 8-oxodG con-tent in pcDNA, from 2.7 6 0.3 8-oxodG/105dG innormal undamaged plasmids to 151.4 6 11 8-oxodG/105dG in damaged plasmids (determined in four sampleseach). Thus, the damaged pcDNA contained approxi-mately 7 base lesions per plasmid.

For the detection of DNA BER activity, we firsttested the ability of mitochondrial extracts from adult braincortex to incorporate [a-32P]dGTP into 8-oxodG-containing pcDNA plasmids. CsCl gradient centrifugation-purified undamaged pSPORT1 plasmids served as negativecontrols. Plasmids were linerarized with EcoRI digestionbefore agarose gel electrophoresis, as previously suggested(Satoh and Lindahl, 1994). Figure 2b shows an ethidiumbromide (EB)-stained gel of pcDNA and pSPORT1, in-dicating equal loading of samples. Four hours of incu-bation of oxidatively damaged pcDNA plasmids with

228 Chen et al.

undamaged pSPORT1 in repair buffer without mito-chondrial protein (or with 60 mg of protein that had beendenatured by heating to 96°C for 5 min prior to reaction)resulted in no incorporation of [a-32P]dGTP (Fig. 2a,lanes 1, 2). Addition of as little as 8 mg of mitochondrialprotein caused detectable radiolabel. Increasing amountsof protein, up to 60 mg, resulted in progressively increas-ing radiolabel (Fig. 2a, lanes 3–7). The relative BERactivity as a function of protein concentration is illustratedin Figure 2c. In all three experiments performed, incor-poration of radiolabeled nucleotides into undamagedpSPORT1 plasmids was negligible, suggesting that theradiolabel detected in pcDNA plasmids is due to lesion-dependent repair.

We then determined the relationship between repairincubation time and the amount of radiolabeled nucleo-tide incorporation into lesioned pcDNA. Lesioned plas-mids were incubated with or without 40 mg of corticalmitochondrial protein in repair buffer for 1, 2, 4, 6, 8, or24 hr. Incubation without protein extracts did not result inany [a-32P]dGTP incorporation (Fig. 3a, lane 1). Onincubation with brain mitochondrial protein, radiolabelincreased with increasing time of incubation up to 8 hr

(Fig. 3a, lanes 2–6). Longer incubation time (24 hr) didnot result in additional incorporation (Fig. 3a, lane 7). Therelative BER activity as a function of repair-incubationtime is presented in Figure 3c. Figure 3b shows the EB-stained agarose gel of Figure 3a.

Shorter incubation time (1–4 hr) had no effect onthe mobility of the plasmids, but incubation for 6–24 hrresulted in slight degradation of the plasmid DNA, whichwas demonstrated as nonspecific radioactive smears (Fig.3a, lanes 5–7).

Excision Activity for Oxidative DNA Lesions inBrain Mitochondrial Extracts

As the first step in the DNA BER pathway, excisionof DNA base lesions requires the activity of at least tworepair enzymes, a specific glycosylase that can recog-nize and cleave the damaged bases, and an apurinic/apyrimidinic (AP) endonuclease that excises the sugar-phosphate backbone of the damaged nucleotide (Dianov

Fig. 1. Determination of contamination of nuclear (PARP and PCNA)or cytosolic protein (b-actin) in mitochondrial protein extracts. West-ern blot analysis was performed using whole cell extracts (Wc) andmitochondrial extracts (Mt) with the amounts of protein as indicated.Note that the mitochondrial marker protein cytochrome c and cyto-chrome c oxidase subunit IV but not any of the cytosolic or nuclearproteins tested are present in mitochondrial preparations. PARP, poly-(ADP-ribose)polymerase; PCNA, proliferating cell nuclear antigen.

Fig. 2. Protein concentration-dependent DNA repair incorporation bymitochondrial extracts from cerebral cortical tissues. The 8-oxodG-containing pcDNA plasmids and undamaged pSPORT1 plasmids wereincubated with various amounts of mitochondrial protein in repairbuffer for 4 hr and linearized by EcoR I restriction before gel electro-phoresis. a: Representative autoradiograph shows [32P]GMP incorpo-ration into pcDNA plasmids in the presence of different amounts ofprotein but not in the absence of protein or in the presence ofdenatured protein (dP). Radiolabel in the undamaged pSPORT1 plas-mids is minimal under each condition. b: Ethidium bromide (EB)-stained gel of a. c: Relative levels of repair incorporation by differentamounts of mitochondrial protein, determined by optical density mea-surement on autoradiograms. Data are reported as mean 6 SEM fromthree independent experiments.

DNA Repair in the Brain Mitochondria 229

and Lindahl, 1994). To examine the activity of these tworepair enzymes in brain mitochondrial extracts, we haveperformed in vitro excision assays. Figure 4a shows thesequences of the two oligodeoxynucleotides used in theexcision assay. We first determined the ability of brainmitochondrial extracts to recognize and specifically cleave8-oxodG in double-stranded DNA. The 21-mer oligonu-cleotide containing 8-oxodG at position 11 was 59-end-labeled with [g-32P]dATP. Complementary oligos wereannealed to form DNA duplexes. Incubation of mito-chondrial extracts with the DNA duplexes for 4 hr re-sulted in a protein concentration-dependent incision ofthe radiolabeled oligomer, generating a specific 10-merproduct (Fig. 4b, lanes 2–6). This cleavage product wasconfirmed by the reaction of Fpg protein (Fig. 4, lane 7),an enzyme known specifically to cleave 8-oxodG. Wethen determined the time-dependent incision of 8-oxodGoligomer by mitochondrial extracts. As expected, incisionproducts were increased with increasing time up to ap-proximately 8 hr of incubation (data not shown).

The substrate preferences of brain mitochondrial8-oxodG glycosylase activity were studied using single-

stranded oligonucleotide containing 8-oxodG or DNAduplexes with 8-oxodG opposite A, C, G, T, as suggestedby others (Croteau et al., 1997). Single-stranded oligomerwas not cleaved by brain mitochondrial protein regardlessof the presence of 8-oxodG (Fig. 4c, lanes 1, 2). Brainmitochondrial extracts had a much greater preference for8-oxodG:C base pairs than 8-oxodG:T, G, or A (Fig. 4c,lanes 3–6). These results are consistent with findings re-garding rat liver mitochondrial 8-oxodG glycosylase ac-tivity, as previously reported (Croteau et al., 1997).

For the detection of brain mitochondrial AP endo-nuclease activity, the standard AP endonuclease assay was

Fig. 3. DNA repair incorporation as a function of incubation time. The8-oxodG-containing pcDNA plasmids were incubated with equalamounts of brain mitochondrial extracts (40 mg) for various periods oftime. a: Representative autoradiograph shows [32P]GMP incorporationinto pcDNA plasmids after 0–24 hr of repair reaction. b: EB-stainedagarose gel of a. c: Relative levels of repair incorporation after variousincubation periods. Data are mean 6 SEM from three independentexperiments.

Fig. 4. Detection of 8-oxodG glycosylase activity in brain mitochon-dria. a: Sequences of the DNA substrate for oligonucleotide incisionassay. The top strand oligonucleotide containing 8-oxodG at position11 was 59-end labeled with [g-32P]ATP before it was annealed to thecomplementary oligonucleotide. b: Protein concentration-dependent8-oxodG glycosylase activity. Various amounts of mitochondrial pro-tein (mtP) were incubated with the radiolabed oligonucleotide duplexin incision assay buffer for 4 hr, generating the specific 10-mer cleavageproduct. Fpg protein (0.5 unit) served as a positive control. c: Substratepreference of brain mitochondrial 8-oxodG glycosylase activity. Mito-chondrial protein (20 mg) was incubated with various 59-end-labeledDNA substrates in incision assay buffer for 3 hr. The brain enzymeactivity did not cleave single-strand oligonucleotide containing eitheran unmodified dG (sG, lane 1) or an 8-oxodG at position 11 (sOG,lane 2). The enzyme cleaves double-strand DNA containing 8-oxodGbut has a greater preference for 8-oxodG:dC base pair (lane 4) than8-oxodG:dA (lane 3), dG (lane 5), or dT (lane 6).

230 Chen et al.

performed as previously described (Pinz and Bogenhagen,1998). The 21-mer oligodeoxynucleotide containing anAP site at position 11 (sequence shown in Fig. 5a) was59-end-labeled with [g-32P]dATP and annealed to com-plementary oligonucleotide to form DNA duplexes. In-cubation of mitochondrial extracts with the DNA du-plexes for 2 hr resulted in a complete incision of theradiolabeled oligomer (0.1 pmol; data not shown). Wesubsequently shortened the incubation time to 20 min,and a protein concentration-dependent AP site incisionreaction was detected (Fig. 5b).

We have determined the 39-end cleavage reactionproducts produced by brain mitochondrial extracts. TheDNA duplexes containing AP sites were incubated withmitochondrial extracts, or bacterial endonuclease III, en-donuclease IV, or Fpg protein, under the standard APendonuclease assay conditions. A representative autoradio-graph showing the 10-mer products generated by the

reaction of various nuclear enzymes is presented in Figure5c. Mitochondrial extracts produced a 10-mer productwith mobility identical to that of endonuclease IV, sug-gesting a 39-OH terminus. These results suggest that brainmitochondria contain a class II AP endonuclease activitythat is able to generate active ends for DNA synthesis byDNA polymerases.

DNA Polymerase Activity in BrainMitochondrial Extracts

DNA polymerization is a key step in the BER path-way for the repair of oxidative DNA lesions. This steprequires a DNA polymerase to fill in the strand breaksgenerated by excision enzymes and synthesize one to fivenew nucleotides (Friedberg, 1985). To determine DNApolymerase activity in brain mitochondria, the standardpolymerase assay was performed. Mitochondrial extractswere able to catalyze the incorporation of 32P-labeledCMP into the M13 ssDNA-M13/PUC duplex in a pro-tein concentration-dependent manner (Fig. 6a).

DNA polymerase-g is the only mitochondrial DNApolymerase identified in mammalian cells so far (Pinz etal., 1995). To determine whether this also represents themajor DNA polymerase activity in brain mitochondria,we have performed DNA polymerase inhibition ex-periments. Three DNA polymerase inhibitors weretested. These included aphidicolin, an inhibitor of DNApolymerase- a, -d, and -e (Mirzayans et al., 1994); thespecific DNA polymerase-g inhibitor N-ethylmaleimide(NEM); and the DNA polymerase-g and -b inhibitorddNTP (in this case, ddCTP). The repair activity ofmitochondrial protein was not affected by aphidicolin(data not shown). Both NEM and ddCTP markedly in-hibited mitochondrial protein-mediated DNA repairsynthesis (Fig. 6a), being consistent with a DNApolymerase-g activity in the mitochondria.

To determine whether DNA polymerase-g repre-sents the predominant DNA polymerase activity in theoverall BER process, inhibition experiments were furthercarried out using in vitro DNA incorporation repair assay.Consistently with the results from the DNA polymeriza-tion assay, NEM and ddNTP (in this case, ddGTP)blocked mitochondrial protein-mediated repair incorpo-ration in 8-oxodG-containing pcDNA plasmids (Fig. 6b).

We have also performed the primer extension assayto detect mitochondrial DNA polymerase activity. Thesequences of the oligonucleotides used in this assay arepresented in Figure 6c. In the absence of dTTP in thereaction mixture, brain mitochondrial extracts resulted ina four-nucleotide extension (from 26-mer to 30-mer) in aprotein concentration-dependent manner (Fig. 6d, lanes1–4), indicating that the extracts are able to perform shortpatch repair. Incubation without mitochondrial proteinshowed no extension (Fig. 6d, lane 5).

DNA Ligase Activity in BrainMitochondrial Extracts

We have performed two assays to detect DNA ligaseactivities in brain mitochondrial extracts. The first assay

Fig. 5. Detection of AP endonuclease activity in brain mitochondria.a: Sequences of the DNA substrate for the oligonucleotide incisionassay. The top strand oligonucleotide containing AP site at position 11was 59-end labeled with [g-32P]ATP before it was annealed to thecomplementary oligonucleotide. b: Protein concentration-dependentAP endonuclease activity. Various amounts of mitochondrial protein(mtP) were incubated with the radiolabed oligonucleotide duplex for20 min, generating the specific 10-mer cleavage product. EndonucleaseIII (endo III, 5 units) served as a positive control. c: Analysis of the39-end cleavage products. The reference enzymes Fpg protein, endo-nuclease III (endo III), and endonuclease IV (endo IV) are known togenerate 39-end phosphate (P), unsaturated sugar, and hydroxyl group(OH), respectively. The mobility of the product from mitochondrialextracts (mtP) is consistent with a hydroxyl group.

DNA Repair in the Brain Mitochondria 231

Fig. 6. Detection of DNA polymerase activity in brain mitochondria.a: Various amounts of mitochondrial protein were incubated with theM13/M13 PUC-primer duplex in the presence of [a-32P]CTP for 30min under standard DNA polymerase assay conditions (see Materialsand Methods). Incorporation of [32P]CMP into the DNA substrateswas measured in the acid-insoluble fraction. The levels of DNA poly-merase activity were protein concentration-dependent. The activitywas inhibited in the presence of NEM or ddCTP. Data are mean 6SEM from three independent experiments. *P , 0.05 vs. inhibitor-freecontrols (ANOVA and post hoc Bonferroni/Dunn tests). b: Overallmitochondrial base excision repair activity was inhibited by the DNApolymerase-g inhibitors NEM and ddGTP. The in vitro repair incor-

poration assays were performed using equal amounts of mitochondrialprotein (30 mg) for 4 hr. Top, autoradiographs showing [32P]GMPincorporation into 8-oxodG-containing pcDNA plasmids in the pres-ence or absence of inhibitors. Bottom, EB staining of the gels on top.c,d: Nucleotide extension assay shows the ability of mitochondrialprotein in performing short patch repair. The sequences of the repairsubstrates for this assay are shown in c. Various amounts of mitochon-drial protein (mtP) were incubated for 30 min with the DNA duplex inwhich the 26-mer oligonucleotide was 59-end labeled. In the absenceof dTTP, the 26-mer was maximally extended for four nucleotides.The short patch repair enzyme DNA polymerase-b (b-pol) served as apositive control.

232 Chen et al.

was to detect the mechanism-based adenylation of DNAligase. A [32P]AMP-ligase complex with a molecularweight between 120 and 140 kDa was detected after themitochondrial extracts were incubated with [a-32P]ATP(Fig. 7a). In the second assay employing specific DNAligation substrates, brain mitochondrial extracts showed aprotein concentration-dependent ligase activity that re-sulted in specific oligo(dT) ligation products (Fig. 7b).

DISCUSSIONThis report demonstrates for the first time that mi-

tochondria of adult rat brain cells possess DNA BERactivity for specific oxidative DNA lesions. The majorfindings from the study are 1) brain mitochondrial proteinextracts are able to carry out BER on naked DNA con-taining defined types of oxidative lesions and 2) brainmitochondrial protein extracts contain all enzyme activi-

ties that are necessary for the BER pathway. These resultsstrongly support the concept that active DNA repair existsin mitochondria of mammalian cells (Croteau and Bohr,1997).

It was a generalized notion until recently that there isno DNA repair mechanism in mitochondria. This resultedfrom a number of early studies showing that certain DNAdamage, such as pyrimidine dimers, was not repaired inmammalian mitochondria (Clayton et al., 1974; LeDouxet al., 1993), whereas it was normally processed in thenucleus through the nucleotide excision repair (NER)pathway. Moreover, repair of mtDNA damage resultingfrom exposure to cisplatin, nitrogen mustard, N-methyl-N-nitro-N-nitrosoguanidine, or other common DNA-damaging agents that are known to induce DNA adductsrequiring NER was also absent (Singh and Maniccia-Bozzo, 1990). More recent studies, however, clearly showthat mammalian mitochondria are able to repair varioustypes of DNA damage through the BER pathway (Cro-teau and Bohr, 1997). At least two lines of evidencesupport the concept of active DNA repair in the mito-chondria. First, oxidative DNA damage and monofunc-tional alkylating agent-induced alkylated base modifica-tions or spontaneous base loss were found to be quicklyremoved from mammalian mtDNA (Satoh et al., 1988;Pettepher et al., 1991; Driggers et al., 1993, 1997; LeDouxet al., 1993; Anson et al., 1998). Second, several enzymesinvolved in BER have been partially purified from mam-malian mitochondria. For example, Croteau et al. (1997)partially purified from rat liver a mitochondrial oxidativedamage-specific glycosylase/endonuclease that has dualfunctions for the recognition and incision of 8-oxodG andAP sites. A mitochondrial endonuclease activity specificfor AP sites in DNA has also been identified in a mousecell line (Tomkinson et al., 1988). However, studies char-acterizing mitochondrial repair enzymes have so far beencarried out in scattered cell types and in different species.Several studies suggest that functionally homologous butnot identical enzymes may be responsible for mtDNArepair in different cell types (Bessho et al., 1993; Croteauet al., 1997; Rosenquist et al., 1997). Accordingly, preciseinformation regarding mtDNA repair in the central ner-vous system cannot be simply derived from previous stud-ies in liver or other nonneuronal tissues.

Brain neurons are highly energetic cells with a highrate of production of DNA-damaging ROS in mitochon-dria (Floyd and Carney, 1992; Chan, 1996). Neurons arepostmitotic cells and, upon damage, they cannot be res-cued by further cell division. Moreover, brain neuronshave an extremely slow turnover of mtDNA during theirrelatively long life span (Wang et al., 1997). These prop-erties may cause neurons to rely heavily on endogenousmechanisms to protect mitochondria from oxidative dam-age, including damage to their genome. The first line ofdefense is known to be an elaborate antioxidant systemdedicated to reducing the levels of ROS (Halliwell, 1989;Murakami et al., 1998). The second line of defense onwhich mitochondria rely is presumably an efficient repair

Fig. 7. Detection of DNA ligase activity in brain mitochondria. a:DNA ligase activity was detected by measuring mechanism-based ad-enylation of the enzyme. Various amounts of mitochondrial protein(mtP) were incubated in DNA ligation buffer with 2 mCi of[a-32P]ATP for 30 min. The arrow indicates the formation of the[32P]AMP-ligase complex. T4 DNA ligase served as a positive control.b: Detection of DNA ligase activity using the oligonucleotide ligationassay. Various amounts of mitochondrial protein (mtP) were incubatedin ligation buffer with duplex of poly(rA)100: oligo(dT)18 for 1 hr.Oligo(dT)18 was 59-end labeled with [g-32P]ATP before annealing topoly(rA)100. This reaction generated the 36- (18 3 2)- or 54-mer (18 33)-specific ligation products.

DNA Repair in the Brain Mitochondria 233

system that can quickly repair oxidative DNA lesions.Here, we provide direct evidence that brain mitochondriaposses such capacity.

The data presented here demonstrate that proteinextracts from brain mitochondria are able to repair thecommon oxidative lesion 8-oxodG in naked plasmidDNA. The DNA repair synthesis initiated by mitochon-drial protein, as indicated by the incorporation of radio-labeled nucleotides into lesioned DNA, shows a proteinconcentration-dependent and repair duration-dependentmanner (Figs. 2, 3). This repair activity is clearly lesion-provoked; incubation with undamaged DNA results innegligible incorporation. These results are similar to thoseof previously reported studies showing the repair ofH2O2-induced oxidative DNA lesions by mitochondriallysates from Xenopus laevis oocytes (Ryoji et al., 1996).Insofar as oxidative DNA damage is known to be repairedpredominantly via the BER pathway, these observationsstrongly suggest that mitochondria contain BER activity.

As previously characterized, the BER process inmammalian cells generally consists of four steps (Dianovand Lindahl, 1994). In the first step, oxidative base damageor a modified base is recognized and removed by theaction of a specific DNA glycosylase, resulting in an abasicsite referred to as the AP site. Second, the DNA sugar-phosphate backbone on the 59-side and the 39-terminalunsaturated aldehyde of an AP site are cleaved by APendonuclease, generating gaps in the strand. Then, a DNApolymerase fills in these gaps and resynthesizes a single, orup to five, new nucleotides. Finally, the ends between thenewly synthsized nucleotides and the adjacent preexistingnucleotides are sealed by a DNA ligase. In the presentstudy, we have performed various assays to examine theenzymatic activities necessary for BER in brain mitochon-dria. Using the oligonucleotide excision assay, we con-firmed that brain mitochondria contain excision enzymesspecific for 8-oxodG and AP site (Figs. 4, 5). A uracilDNA glycosylase activity was also detected (data notshown). By studying time-dependent incision, we foundthat the process for cleavage of the AP site is much fasterthan that for 8-oxodG or uracil. In this regard, the actionby DNA glycosylases is likely to be a rate-limiting factor inmitochondrial BER. Because the excision assays wereperformed using crude mitochondrial protein extracts, weare not able to resolve the 39- or 59-end products gener-ated by the 8-oxodG or uracil glycosylase. It has beenreported that the rat liver 8-oxodG glycosylase contains anadditional AP endonuclease-like activity and produces a39-deoxyribose moiety and a 59-phosphate group via theb-elimination mechanism (Croteau et al., 1997). Theseactions are distinctive from that of nuclear glycosylasesidentified in bacterial or mammalian cells (Bessho et al.,1993; van der Kemp et al., 1996). Using the AP siteexcision assay, however, we have resolved the 39-endproduct generated by brain mitochondrial extracts andidentified a hydroxyl nucleotide residue (Fig. 5c). Thus,brain mitochondrial extracts are able to provide activeprimer termini for DNA repair synthesis. This result is

consistent with an AP endonuclease class II action com-patible with that of the mitochondrial AP endonucleasepreviously found in a mouse cell line (Tomkinson et al.,1988). Whether the generation of 39-end hydroxyl resi-dues by brain mitochondrial extracts is due to the APendunuclease activity alone or combined with an exonu-clease activity is unclear. Nevertheless, a mitochondrialexonuclease activity that is independent of AP endonu-clease has so far not been identified in mammalian cells.

We have identified a DNA polymerase activity inbrain mitochondrial extracts, which has some characteris-tics of DNA polymerase-g as suggested by others (Longleyet al., 1998). For example, either NEM or ddNTP, butnot aphidicolin, can block DNA repair synthesis by mi-tochondrial extracts. Moreover, as shown by the primerextension assay (Fig. 6d), this DNA polymerase activity iscapable of short patch repair. Thus, mitochondria in adultbrain neurons appear to employ DNA polymerases ho-mologous to, but not identical to, that in nucleus for theBER process, insofar as DNA polymerase-b is thepredominant form found in the nucleus (Subba Raoet al., 1985). Compared to DNA polymerase-b, DNApolymerase-g is a highly processive enzyme, possesses aproofreading 39- to 59-exonuclease activity, and thus pre-sumably has a lower error rate (Pinz et al., 1995). Furtherinvestigations are necessary to characterize the detail func-tional properties, especially the proofreading capacity foroxidative lesions of this enzyme in the brain mitochondria.

Whereas a variety of nuclear DNA ligases have beendiscovered in mammalian systems (Lindahl and Barnes,1992), little is known concerning mitochondrial DNAligase. Levin and Zimmerman (1976) previously reporteda DNA ligase activity from mitochondria of rat liver, butthe enzyme was not characterized. More recently, Pinzand Bogenhagen (1998) characterized a mitochondrialDNA ligase in Xenopus laevis that shares functional andamino acid sequence similarity with the human nuclearDNA ligase III. In the present study, we found that brainmitochondrial extracts can form an enzyme-adenylate in-termediate and are able to ligate oligo(dT) strands annealedto poly(rA) (Fig. 7). These results indicate that brainmitochondria contain a specific DNA ligase activity.Given the fact that DNA ligase plays a crucial role insealing DNA nicks and is essential for the completion ofBER, these results warrant further characterization of thisenzymatic activity.

The results from this research have clear significance.This study has, for the first time, identified DNA repairactivity in adult brain mitochondria. This activity mayrepresent an important endogenous cellular protectivemechanism against oxidative stress, which should be par-ticularly relevant to the highly energetic brain cells. Fur-thermore, this study has partially resolved the BER processin the brain mitochondria. These established methods forthe detection of repair enzyme activities will be valuablefor future investigations in determining whether and howalterations in mitochondrial DNA repair activity may con-tribute to the pathogenesis of various neurological disor-

234 Chen et al.

ders involving mtDNA damage and mitochondrial dys-function.

ACKNOWLEDGMENTSThis work was supported by NIH/NINDS grants

NS 36736 and NS 38560 to J.C. J.C. was also supportedin part by the Geriatric Research, Education and ClinicalCenter, Veterans Affairs Pittsburgh Health Care System,Pittsburgh, Pennsylvania.

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