age-dependent decline of dna repair activity for oxidative lesions in rat brain mitochondria

Age-dependent decline of DNA repair activity for oxidative lesions

in rat brain mitochondria

Dexi Chen,*,� Guodong Cao,*,� Teresa Hastings,*,� Yiqin Feng,*,�Wei Pei,*,� Cristine O’Horo*,�and Jun Chen*,�,�

*Department of Neurology, �Pittsburgh Institute for Neurodegenerative Disorders, University of Pittsburgh School of Medicine,

Pittsburgh, Pennsylvania, USA

�Geriatric Research, Educational and Clinical Center, Veterans Affairs Pittsburgh Health Care System, Pittsburgh, Pennsylvania,

USA

Abstract

Endogenous oxidative damage to brain mitochondrial DNA

and mitochondrial dysfunction are contributing factors in aging

and in the pathogenesis of a number of neurodegenerative

diseases. In this study, we characterized the regulation of

base-excision-repair (BER) activity, the predominant repair

mechanism for oxidative DNA lesions, in brain mitochondria

as the function of age. Mitochondrial protein extracts were

prepared from rat cerebral cortices at the ages of embryonic

day 17 (E17) or postnatal 1-, 2-, and 3-weeks, or 5- and

30-months. The total BER activity and the activity of essential

BER enzymes were examined in mitochondria using in vitro

DNA repair assay employing specific repair substrates.

Mitochondrial BER activity showed marked age-dependent

declines in the brain. The levels of overall BER activity were

highest at E17, gradually decreased thereafter, and reached

to the lowest at the age of 30-month (�80% reduction). The

decline of overall BER activity with age was attributed to the

decreased expression of repair enzymes such as 8-OHdG

glycosylase and DNA polymerase-c and, consequently, the

reduced activity at the steps of lesion-base incision, DNA

repair synthesis and DNA ligation in the BER pathway. These

results strongly suggest that the decline in BER activity may

be an important mechanism contributing to the age-dependent

accumulation of oxidative DNA lesions in brain mitochondria.

Keywords: 8-hydroxyl-2¢-deoxyguanosine, aging, DNA

damage, mitochondrial dysfunction, oxidative stress.

J. Neurochem. (2002) 81, 1273–1284.

Mitochondrial DNA (mtDNA) encodes 13 functional

peptides, all of which directly participate in mitochondrial

oxidative phosphorylation and ATP production, and are

crucial for the cell to maintain its normal homeostasis. Since

mtDNA is localized in an oxygen-rich environment and lacks

protection by histones, mtDNA is particularly susceptible to

and indeed constantly damaged by reactive oxygen species

(ROS) that are generated as by-products of the electron

transport chain (Ames et al. 1993). Excessive accumulation

of endogenous oxidative damage to brain mtDNA and the

decline of mitochondrial respiratory function have long been

implicated in the aging process (Miquel et al. 1980; Bowling

et al. 1993; Shigenaga et al. 1994; Hudson et al. 1998; Wei

et al. 1998; Barja and Herrero 2000; Morre et al. 2000).

There is strong evidence that several forms of oxida-

tive mtDNA damage, including 8-oxo-2¢-deoxyguanosine(8-oxodG), deletions and circular dimers, are markedly

increased in aged human brain (Bulpitt and Piko 1984;

Cortopassi et al. 1992; Mecocci et al. 1993), especially in

regions that have a higher metabolic rate (Corral-Debrinski

et al. 1992; Cortopassi et al. 1992; Zhang et al. 1992). It has

been suggested that unrepaired oxidative mtDNA damage

may affect normal mitochondrial gene expression and thus

cause defective electron transfer in complexes I–IV of the

Received February 12, 2002; revised manuscript received March 6,

2002; accepted March 7, 2002.

Address correspondence and reprint requests to Dr Jun Chen,

Department of Neurology, S-507, Biomedical Science Tower, University

of Pittsburgh School of Medicine, Pittsburgh, PA 15213, USA.

E-mail: [email protected]

Abbreviations used: a-pol, polymerase a; AIF, apoptosis-inducingfactor; AP, apurinic/apyrimidinic; APE, AP endonuclease; BER,

base-excision-repair; BSA, bovine serum albumin; Cox-IV, cyto-

chrome c oxidase IV; cyto c, cytochrome c; DTT, dithiothreitol; E17d,

embryonic day 17; mtDNA, mitochondrial DNA; Ogg1, 8-oxodG

glycosylase; 8-oxodG, 8-oxo-2¢-deoxyguanosine; PMSF, phenyl-

methylsulfonyl fluoride; Pxm, postnatal x month; Pxw, postnatal x week;

ROS, reactive oxygen species; SDS, sodium dodecyl sulfate; UDG,

uracil DNA glycosylase.

Journal of Neurochemistry, 2002, 81, 1273–1284

� 2002 International Society for Neurochemistry, Journal of Neurochemistry, 81, 1273–1284 1273

respiratory chain (Harmon et al. 1987; Bowling et al. 1993);

this may lead to further increases in the production of ROS,

perpetuating the cycle of oxidative damage to mitochondria

(Miquel et al. 1980; Wei et al. 1998). This mitochondrial

theory of cell aging may have significant relevance in the

pathogenesis of a number of age-related neurological

disorders (Parker et al. 1990; Shoffner et al. 1991; Corral-

Debrinski et al. 1992; Swerdlow et al. 1996).

The specific mechanisms for the accumulation of oxidative

mtDNA damage in aged brain are not known, but it is likely

due to either an increased rate of damage formation over time

or an age-dependent decline in DNA repair capacity in the

mitochondria. The DNA base excision repair (BER) pathway

is known to be the predominant mechanism for the repair of

oxidative DNA lesions in mammalian cells. However,

whether the BER machinery exists in mammalian mitochon-

dria as in the cell nucleus has been a matter of controversy

until recently (Croteau and Bohr 1997). With the new

development of various in vitro repair assays and the

availability of specific substrates for these assays, it has

now been confirmed that active BER exists in mammalian

mitochondria (Croteau et al. 1997; Rosenquist et al. 1997;

Pinz and Bogenhagen 1998), including brain mitochondria

(Chen et al. 2000). Moreover, it has been reported that

mammalian mitochondria are able to repair various forms of

endogenous oxidative DNA damage, such as base damage,

single-strand breaks and apurinic/apyrimidinic sites, likely

via the BER pathway (Taffe et al. 1996; Driggers et al. 1997).

Accordingly, BER may play a critical role in maintaining the

integrity of mtDNA, in which oxidative lesions are constantly

generated throughout the life span of the cell.

In the present study, we investigated the age-dependent

regulation of BER activity in rat brain mitochondria using

assays that detect either the overall BER activity or

individual enzymatic activities along the BER pathway. We

aimed to address the hypothesis that the age-dependent

accumulation of endogenous oxidative mtDNA damage may

be at least in part due to the decline of BER activity in the

mitochondria.

Materials and methods

Isolation and purification of brain mitochondria

All animal procedures were performed using protocols approved by

the Animal Care and Use Committee at the University of Pittsburgh

and in accordance with the principles outlined in the National

Institutes of Health Guide for the Care and Use of Laboratory

Animals. In the present study, experiments were done using three

different sets of Sprague–Dawley rats of the following ages: 17-day-

old embryos (E17), postnatal 1 week (P1w), postnatal 2 week

(P2w), postnatal 3 week (P3w), postnatal 5 month (P5m), and

postnatal 30 month (P30m). The P30m rats were purchased from the

National Institute of Aging, and the rest from Hilltop Sprague–

Dawley (Scottsdale, PA, USA).

All procedures for the isolation and purification of brain

mitochondria were carried out at 4�C as previously described (Sunand Gilboe 1994; Ryoji et al. 1996), with slight modifications

(Chen et al. 2000). To minimize the experimental variations

among different ages due to isolation procedures, all brains within

the same set were processed at the same time. Briefly, brain

cortical tissues taken from the frontal-parietal cortex, approxi-

mately 1 g per sample, were minced and homogenized using a

Dounce homogenizer in the M-SHE buffer containing 0.21 M

mannitol, 0.07 M sucrose, 10 mM HEPES–KOH at pH 7.4, 1 mM

EDTA, 1 mM EGTA, 0.15 mM spermine, and 0.75 mM spermidine.

The following protease inhibitors were added immediately before

use: 1 mM dithiothreitol (DTT), 1 mM phenylmethylsulfonyl

fluoride (PMSF), and 1 lg/mL each of leupeptin, aprotinin and

pepstatin A. After lysis for 30 min on ice, unbroken cells and

nuclei were pelleted at 1200 g. The supernatant, containing the

mitochondria, was centrifuged at 10 000 g for 15 min to pellet the

mitochondria. The mitochondrial pellet was resuspended in a

solution containing 3% Ficoll 400, 0.12 M mannitol, 0.03 M

sucrose, and 25 lM EDTA (pH 7.4) and gently layered twice in

6% Ficoll 400 solution to produce a discontinuous density

gradient. After centrifugation at 10 400 g for 25 min, the sediment

was resuspended in 1 · M-SHE buffer containing 3 mg/mL

digitonin for 15 min. This was followed by centrifugation at

10 500 g for 15 min. The pellet was washed with 1 · M-SHE andthen lysed for 30 min in a lysis buffer containing 20 mM HEPES

(pH 7.4), 400 mM KCl, 1 mM EDTA, 5% glycerol, 0.5% Triton-X

100, 2 mM DTT, 0.5 mM PMSF, and 1 lg/mL each of aprotinin,pepstatin A and leupeptin. The lysate was centrifuged at

130 000 g for 1 h, concentrated to 5–10 mg/mL protein in a

speed vacuum vaporizer (model SC100; Savant Inc., Farmingdale,

NY, USA), and stored at ) 80�C until use. Under this condition,

the protein lysate remains stable for a minimum of 6 months.

To determine the purity of the Ficoll-400 gradient-purified

mitochondrial protein, immunoblotting analysis was performed to

ascertain the levels at which mitochondrial protein was contamin-

ated with cytosolic or nuclear proteins. The procedures were the

same as described previously (Chen et al. 2000). The following

markers were used: cytosolic protein, b actin; nuclear proteins,

PARP and histone; mitochondrial proteins, cytochrome c, cyto-

chrome c oxidase, and apoptosis-inducing factor (AIF).

In vitro DNA incorporation repair assay

This assay examined the ability of mitochondrial protein extracts to

incorporate 32P-dGTP into oxidatively damaged plasmids (Chen

et al. 2000; Nagayama et al. 2000). The DNA repair substrate used

in the present study was a pcDNA plasmid (Stratagene) containing

the oxidative adduct 8-oxodG, a common DNA lesion known to be

repaired by the BER pathway. These experiments provided an

estimation of overall BER activity in the mitochondria.

The DNA repair substrate was prepared using photoactivated

methylene blue (MB) to induce 8-oxodG in purified pcDNA

plasmids as described previously (McBride et al. 1992; Chen et al.

2000). The content of 8-oxodG in the plasmids was verified using

HPLC-EC. Typically, under the described conditions (Chen et al.

2000), incubation of MB for 30 min induced approximately 150

8-oxodG/510dG in the plasmids, representing an approximately

50-fold increase over the baseline levels (Chen et al. 2000).

1274 D. Chen et al.

� 2002 International Society for Neurochemistry, Journal of Neurochemistry, 81, 1273–1284

To perform the repair assay, mitochondrial extracts (amounts of

protein as indicated in the experimental protocol) were incubated for

a period of time as indicated in the protocol at 32�C in 50 lL ofreaction mixtures containing 0.3 lg each of the 8-oxodG-rich

plasmids, 45 mM HEPES–KOH (pH 7.8), 70 mM KCl, 5 mM

MgCl2, 1 mM DTT, 0.4 mM EDTA, 2 mM ATP, 20 lM each of

dATP, dTTP, and dCTP, 8 lM dGTP, 2 lCi of [a-32P]GTP (ICN),40 mM phosphocreatine, 2.5 lg creatine phosphokinase, 3% gly-

cerol, 20 lg/mL BSA, 2 mM NAD+, and 1 mM b-mercaptoethanol.In all experiments, undamaged pSPORT1 plasmids served as

negative controls. The reaction was terminated by the addition of

proteinase K (240 lg/mL), sodium dodecyl sulfate (SDS) (to 1%),

and EDTA (to 20 mM) and incubation of the samples for 30 min at

37�C. Plasmid DNAwas phenol-extracted from the mixture, 5 lg ofcarrier tRNA was added, and the DNA precipitate was dissolved in

20 lL of TE buffer. The samples were treated with 10 units of

BamHI (Gibco, BRL, Rockville, MD, USA) overnight at 37�C tolinearize the DNA, and then separated by electrophoresis on a 1%

agarose gel. DNA bands on the gel were visualized using UV light

and photographed. Radioactive nucleotide incorporation into the

DNA was detected using autoradiography. Autoradiogram signals

on the films were semiquantified (optical density · area) by a geldensitometric scanning program using the Microcomputer Imaging

Device (MCID) image analysis system (St. Catharine’s, Ontario,

Canada). All densitometric values for DNA radiolabels were

normalized to values for UV photographs of DNA bands on the

same lane.

In vitro oligonucleotide incision assays

The following experiments, including the in vitro oligonucleotide

incision assay, DNA polymerization assay, and DNA ligase assay

were designed to detect the repair activities of mitochondrial protein

extracts at each step along the base excision repair pathway. The

in vitro oligonucleotide incision assay was used to estimate the

ability of mitochondrial protein extracts to recognize and remove

three types of DNA lesions: 8-oxodG, uracil, and apurinic/

apyrimidinic (AP) abasic site. The DNA substrate used in this

assay was a 50-mer oligodeoxynucleotide containing either an

8-oxodG, uracil, or AP site at position 26 (for sequences see Fig. 2).

The oligonucleotide containing either the lesion or normal unmodi-

fied bases was 5¢-end-labeled using T4 polynucleotide kinase and[c-32P]ATP, and the reaction mixture was passed through a G-25spin column (5 prime/3 prime, Inc., Boulder, CO, USA) to remove

the free unlabeled [c-32P]ATP. The labeled oligonucleotide wasthen annealed to the complementary oligonucleotide in 100 mM

KCl, 10 mM Tris (pH 7.8), and 1 mM EDTA by heating the

oligonucleotides to 80�C, and then allowed to cool slowly to roomtemperature. In the case of the oligonucleotide that contained an

AP site, the sample was heated to 55�C instead of 80�C, due to theheat lability of the AP site. The reaction mixture for the incision

assay contained 40 mM HEPES–KOH (pH 7.6), 20 mM KCl, 2 mM

CaCl, 2 mM DTT, 1 mM EDTA, 20 lM zinc acetate, 10% glycerol,

0.05% Triton X-100, 0.1 mg/mL bovine serum albumin (BSA), 200

fmol of 32P-labeled DNA duplex, and mitochondrial protein extracts

at the amount indicated in each experiment. The reaction was carried

out at 37�C for a period of time according to the experimental

protocol, and then terminated by the addition of an equal volume of

loading buffer containing 90% formamide, 0.002% bromophenol

blue, and 0.002% xylene cyanol. The sample was heated to 80�C for2 min and subjected to electrophoresis on a denaturing 20%

polyacrylamide gel containing 7 M urea. After electrophoresis, the

gel was subjected to autoradiography and densitometry analysis.

DNA polymerase assay

The primer extension assay was performed to estimate the specific

DNA polymerase activity in the mitochondrial protein extracts

(Chen et al. 2000). The oligonucleotides used in these studies

(sequences shown in Fig. 4) were custom-made and PAGE-purified

(Biosynthesis, Dallas, TX, USA). The 26-mer oligodeoxynucleotide

was 5¢-end-labeled using T4 polynucleotide kinase and [c-32P]ATP(specific activity, 5 · 106 cpm/pmol) and purified using a G-25 spincolumn. The labeled oligomer was then annealed to the 50-mer

M50S oligonucleotide as described above. The reaction mixture

consisted of 45 mM HEPES–KOH (pH 7.8), 70 mM KCl, 4 mM

MgCl2, 1 mM DTT, 2 mM ATP, 1 mM EDTA, 40 mM phosphocrea-

tine, 2.5 lg creatine phosphokinase, 20 lM each of dATP, dCTP,

and dGTP, 3% glycerol, 20 lg/mL BSA, and 1 mM b-mercapto-ethanol. Mitochondrial extracts were incubated with the annealed

DNA (100 fmol) in the reaction mixture for 50 min at 32�C. Thereaction was terminated by adding 10 lL of loading buffer

containing 90% (v/v) formamide, 0.1% (w/v) bromphenol blue,

and 20 mM EDTA, and then heating at 80�C for 2 min. The

reaction products were separated by electrophoresis on a 15%

polyacrylamide gel containing 7 M urea, and detected using

autoradiography.

DNA ligation assay

DNA ligase activity in mitochondrial protein extracts was examined

using the oligonucleotide ligation assay as previously described

(Pinz and Bogenhagen 1998; Chen et al. 2000). This was done by

incubation of mitochondrial extracts with DNA substrates prepared

by annealing 10 pmol of 5¢-32P-oligo(dT)18)1 lg of poly(rA)100 inreaction mixtures containing DNA ligase buffer and 1 mM ATP,

40 mM phosphocreatine, 2.5 lg creatine phosphokinase. The ligasebuffer consisted of 20 mM Tris (pH 8.0), 40 mM NaCl, 5 mM

MgCl2, 5 mM DTT, 8% glycerol, and 0.02% Triton X-100. The

reaction was continued at 32�C for 1 h. Ligation products were

analyzed by electrophoresis on a 10% polyacrylamide gel contain-

ing 8 M urea and detected by autoradiography.

Combined DNA polymerase and ligase assay

The principle of the assay is shown in Fig. 6(a). Autoradiography of

this assay shows specific repair-products, either the 26-mer

(representing the DNA polymerase activity) or the 50-mer (repre-

senting both DNA polymerase and DNA ligase activities), with high

resolution. To perform the assay, the 50-mer oligonucleotide

(300 fmol) containing a uracil at position 26 (for full sequence,

see Fig. 2) was annealed to the complementary oligonucleotide. The

DNA duplex was subjected to digestion by purified UDG (5 U) and

endonuclease IV (10 U) at 37�C for 15 min in a buffer containing50 mM HEPES–KOH (pH 7.6), 5 mM MgCl2, 50 mM KCl, 0.05%

Triton X-100 and 0.1 mg/mL BSA, and then the mixture was heated

to 55�C for 10 min to inactivate UDG and endonuclease IV. This

reaction produced single-nucleotide nicks in the DNA duplex, which

subsequently served as the repair substrate for DNA polymerase and

DNA ligase. Mitochondrial extract (20 lg) was then incubated with

Mitochondrial DNA repair in brain aging 1275


this repair substrate in the same buffer above with the additions of

2 lCi of [a-32P]GTP, 40 mM phosphocreatine, 2.5 lg creatine

phosphokinase, 3% glycerol, 2 mM NAD+, and 1 mM b-mercapto-ethanol. The reaction was carried out at 32�C for 1 h before it wasterminated by adding an equal volume of loading buffer and heating

to 80�C for 2 min. The reaction products were separated in a 15%polyacrylamide gel containing 7 M urea, and detected using

autoradiography.

Western blot analysis

The levels of essential mitochondrial BER enzymes, 8-oxodG

glycosylase (Ogg1) and DNA polymerase c (c-pol), were examinedin brain mitochondrial extracts of different ages using western blot

analysis. The mitochondrial markers cytochrome c (cyto c), cyto-

chrome c oxidase IV (Cox-IV), and apoptosis-inducing factor (AIF)

were also detected and served as reference proteins. Western blot

analysis was performed using the standard method (Chen et al.

1998). The working dilutions for the following antibodies were per

the manufacturers’ suggestions: c-pol rabbit polyclonal antibody(NeoMarkers, Fremont, CA, USA), Ogg1 rabbit polyclonal antibody

(Novus Biological, Littleton, CO, USA), cyto c monoclonal

antibody (Pharmingen, San Diego, CA, USA), Cox-IV monoclonal

antibody (Molecular Probes, Eugene, OR, USA), and AIF rabbit

polyclonal antibody (Chemicon International, Inc., Temecula, CA,

USA). Immunoreactivity for each protein on each individual lane of

the blots was semiquantified using the Microcomputer Imaging

Device (MCID, St. Catherine’s, Ontario, Canada) image analysis

system (Chen et al. 1998).

Data analysis

All quantitative data are reported as mean ± SEM. Comparisons of

DNA base excision repair activity, oligonucleotide incision activity,

DNA polymerase activity, and DNA ligase activity among different

experimental groups were made using ANOVA and post hoc

Bonferroni/Dunn tests. A level of p < 0.05 was considered

statistically significant.

Results

Purity of the mitochondrial protein preparation

To determine whether the mitochondrial protein extracts

prepared for DNA repair assays were contaminated with

detectable amounts of cytosolic or nuclear proteins, western

blots were performed to detect b-actin, PARP, histone H1,and three mitochondrial markers, cytochrome c, cytochrome

c oxidase, and AIF. Immunoreactivity of cytochrome c,

cytochrome c oxidase, and AIF, but not any of the three

cytosolic or nuclear proteins, was readily detectable in the

mitochondrial protein preparations (data not shown).

Fig. 1 Age-dependent decline of overall BER activity in mitochondrial

extracts. The DNA repair incorporation assay was performed using the

8-oxodG-containing pcDNA plasmids and undamaged pSPORT plas-

mids. (a) Control reactions show [32P]GMP incorporation into pcDNA

plasmids in the presence of different amounts of mixed protein, drawn

equally from brains of different ages. (b) Ethidium bromide (EB)-stained

gel of (a). (c) Relative levels of repair incorporation as the function of

protein concentration, determined by optical density measurement on

three autoradiographs. (d) Representative autoradiograph shows the

age-dependent decline in DNA repair incorporation. An equal amount

of protein (20 lg) was used in each reaction. (e) EB-stained gel of d.

(f) Relative levels of repair incorporation as the function of age, deter-

mined on three independent experiments. *p < 0.05 versus E17, P1w,

P2w, or P3w; #p < 0.05 versus E17, P1w, P2w, P3w, or P5m.

1276 D. Chen et al.


Age-dependent decline of overall BER activity

in mitochondria

Figure 1 illustrates the results of in vitro DNA incorporation

repair assays examining the overall BER activity in the

mitochondria. Control experiments were performed using a

protein mixture containing equal amounts of protein drawn

from each age to be tested, which helped determine the

protein concentration to be employed in subsequent assays.

As shown in Fig. 1(a and b), incubation for 1 h of

oxidatively damaged pcDNA plasmids in repair buffer with

mitochondrial protein in the concentration range of 5–40 lg(in 50 lL of reaction mixture) resulted in progressively

increasing amounts of radiolabel in pcDNA (Fig. 1a, lanes

2–5). Increasing the protein to 60 lg failed to further

increase the radiolabel (lane 6). In all three control experi-

ments performed, incorporation of radiolabel nucleotides into

the undamaged pSPORT plasmids was not detectable,

indicating that the radiolabel detected in pcDNA plasmids

is due to lesion-dependent repair.

Mitochondrial protein-initiated radiolabel incorporation

into damaged PcDNA was measured in brain extracts

(20 lg per sample) from rats of different ages (Figs 1d–f).

There was a marked age-dependent decline in the repair

incorporation: E17d showed the highest level of incor-

poration, whereas P1w–P3w showed a moderate decline

(�20–40% decrease compared with E17d), while P5m

(�60% decrease) and especially P30m (�80% decrease)

showed a marked decline in repair incorporation. Statistical

analysis based on three independent experiments using three

different sets of brain samples showed that the levels of P5m

and P30m were significantly lower than each of E17d–P3w

(p < 0.05). Moreover, the difference between P5m and P30m

also reached statistical significance (Fig. 1f).

Age-dependent decline of excision activity for base

lesions

As the first step in the BER pathway, excision of DNA base

lesions requires the action of at least two repair enzymes: a

specific glycosylase that can recognize and cleave the damaged

bases such as 8-oxodG, and an AP endonuclease (APE) that

excises the sugar-phosphate backbone of the damaged

nucleotide. We performed the in vitro excision assays to

Fig. 2 Age-dependent decline of base incision activity in brain

mitochondrial extracts. (a) Sequences of the DNA substrates for

oligonucleotide incision assays. The top strand oligonucleotide con-

taining either uracil or 8-oxodG at position 26 was 5¢-end labeled with

[a-32P]ATP before it was annealed to the complementary oligonu-

cleotide. (b) Representative autoradiograph shows age-dependent

decline in UDG activity in mitochondrial extracts. (c) Representative

autoradiograph shows age-dependent decline in 8-oxodG glycosylase

activity in mitochondrial extracts. In all experiments, equal amount of

DNA substrates (200 fmol) and protein (20 lg) were used in each

reaction, and the 25-bp oligo was the specific cleavage product of

these assays. (d) Quantitative analysis of UDG and 8-oxodG glyco-

sylase activity in mitochondrial extracts as the function of age, deter-

mined by optical density measurements on autoradiographs from three

independent experiments. *p < 0.05 versus E17, P1w, or P2w;

#p < 0.05 versus E17, P1w, P2w, P3w, or P5m.



determine the activity of three excision enzymes, 8-oxodG

glycosylase, uracil DNAglycosylase (UDG) andAPE, in brain

mitochondria of different ages. Figure 2(b and c) shows the

detection of UDG and 8-oxodG glycosylase activities in

mitochondrial protein, respectively. Incubation of mitochond-

rial protein (20 lg) for 1 hwith the radiolabeledDNAduplexescontaining an 8-oxodG or uracil at position 26 resulted in the

incision of the lesion-containing oligomer, generating a

specific 25-mer product. Consistent with the results of the

incorporation assay, both 8-oxodG glycosylase and UDG

activities showed an age-dependent decline in brain mito-

chondrial extracts. In both cases, P30m showed the lowest

levels, significantly lower than each preceding age (p < 0.05).

Brain mitochondria contain relatively high levels of APE

activity for the natural AP site (Chen et al. 2000). In this study,

2 lg of mitochondrial protein was incubated for 10 min withthe radiolabeled DNA duplexes containing a natural AP site at

position 26, generating the specific 25-mer oligomer (Fig. 3a).

The AP site in DNA was produced by treating the uracil-

containing DNA duplexes with purified UDG (see Materials

and methods). As shown in Figs 3(a and b), a significant

decline of APE activity was detected at P5m and P30m as

compared with newborn rats. However, there was no

significant difference in APE activity between E17d and

newborn rats.

Age-dependent decline of mitochondrial DNA

polymerase activity

DNA polymerization is a key step in the BER pathway for

the repair of oxidative DNA lesions. Our previous study

found that DNA polymerase-c is the predominant DNA

polymerase activity in brain mitochondria for DNA repair

synthesis (Chen et al. 2000). In this study, we performed the

primer extension assay to measure mitochondrial DNA

polymerase-c activity. Control experiments showed that, inthe absence of dTTP in the reaction mixture, brain mito-

chondrial protein extracts resulted in a four-nucleotide

extension (from 26-mer to 30-mer) in a protein concentra-

tion-dependent manner (Figs 4a–c), whereas incubation

without mitochondrial protein showed no extension.

DNA polymerase-c activity was then measured in brainmitochondrial protein extracts (20 lg) from rats of different

ages (Figs 4d and e). As compared with E17d and newborn

rats, P5m and P30m showed marked decreases (�80%) inmitochondrial DNA polymerase-c activity. However, no

significant difference was detected between P5m and P30m.

Age-dependent decline of mitochondrial DNA ligase

activity in mitochondria

DNA ligase activity in mitochondrial protein extracts from

rats of different ages was examined using the standard

oligonucleotide ligation assay employing the 32P-oligo

(dT)18/poly(rA)100 DNA duplexes (Chen et al. 2000). This

assay generates the 36-mer (T36), and to a lesser extent, the

54-mer (T54) ligation products (Fig. 5a). As shown in

Figs 5(a and b), DNA ligase activity in brain mitochondria

exhibited a marked age-dependent down-regulation. As

compared with E17d, DNA ligase activity was significantly

decreased in newborn rats (T36, �50–60%) as well as inP5m and P30m rats (> 80%).

To further confirm the age-dependent changes in DNA

polymerase and DNA ligase activities in brain mitochondria,

we performed additional assays based on the cell-free

reconstitution of the BER system using purified enzymes

(Fig. 6). As shown in Figs 6(a and b), the repair substrate

was generated by the purified UDG and endonuclease IV

(produce single-nucleotide nicks in the DNA duplex), which

can be used to detect the capability of cell extracts to

incorporate [32P]GMP into the DNA (requiring DNA

polymerase and ligase). As shown in Figs 6(c and d), the

specific 26-mer (generated by the DNA polymerase activity

alone) and 50-mer (generated by combined DNA polymerase

and DNA ligase activities) repair products showed marked

decreases in the presence of mitochondrial extracts from aged

brains as compared with the newborn and young rats. Being

Fig. 3 Age-dependent decline of AP endonuclease activity in brain

mitochondrial extracts. (a) Equal amount of protein (2 lg) from each

age was incubated with the 5¢-end labeled 50-bp DNA duplex

(100 fmol) containing an AP site at position 26 (see Materials and

methods) at 37�C for 10 min, generating the specific 25-bp cleavage

product. Note that the amounts of the cleavage product was

decreased in adult and aged rats. (b) Quantitative analysis of AP

endonuclease activity in mitochondrial extracts as the function of age,

determined by optical density measurements on autoradiographs from

three independent experiments. *p < 0.05 versus E17, P1w, P2w, or

P3w; #p < 0.05 versus E17, P1w, P2w, P3w, or P5m.

1278 D. Chen et al.


consistent with the results from the primer extension assays

(Fig. 4) and oligonucleotide ligation assays (Fig. 5), the

differences between P5m and P30m did not show statistical

significance.

Age-dependent decline of the expression

of mitochondrial repair enzymes

The levels of the essential BER enzymes Ogg1 and c-polwere examined in brain mitochondrial extracts of different

ages. The expression of both repair enzymes was subjected to

age-dependent down-regulation, which reached the lowest

levels in P30m (Fig. 7). While the level of c-pol was notdifferent between P5m and P30m, the level of Ogg1 was

significantly lower in P30m than each preceding age

(p < 0.05). These patterns of age-dependent changes in

Ogg1 and c-pol were similar to that of the activity assaysdescribed above (Figs 2c and 5).

In contrast to the age-dependent down-regulation of BER

enzymes, the levels of mitochondrial markers cyto c and

Cox-IV were slightly increased in the adult and aged rats,

whereas the levels of AIF remained unchanged with age

(Figs 7a and b).

Discussion

The results from this study have, for the first time, provided

direct evidence that there is a marked age-dependent decline

in BER activity for oxidative DNA lesions in rat brain

mitochondria. Further, the results demonstrate that the

decline of BER activity is attributed to the decreased

(d)(b)

(a)

(c) (e)

Fig. 4 Age-dependent decline of DNA polymerase-c activity in brain

mitochondrial extracts. (a) Sequences of the DNA substrates for the

primer extension assay. The 26-bp oligonucleotide was 5¢-end labeled

before it was annealed to the 50-bp complementary oligonucleotide.

(b) Control reactions show primer extension in the presence of

different amounts of mixed protein, drawn equally from brains of

different ages. In the absence of dTTP, the 26-bp oligonucleotide was

maximally extended for four nucleotides, generating the 30-bp product.

The Klenow-large-fragment of DNA polymerase I enzyme served as

the positive control in this assay (P, lane 1). (c) The levels of primer

extension as the function of protein concentration, determined by

optical density measurements on three autoradiographs. (d) Repre-

sentative autoradiograph shows age-dependent decline in DNA

polymerase-c activity in mitochondrial extracts. Equal amount of DNA

substrate (100 fmol) and protein (20 lg) was used in each reaction.

Note that the amount of the 30-bp extension product was decreased in

the adult and aged rats. (e) Quantitative analysis of DNA polymerase-c

activity in mitochondrial extracts as the function of age, determined by

optical density measurements on autoradiographs from three inde-

pendent experiments. *p < 0.05 versus E17, P1w, P2w, or P3w.



activity of the repair enzymes that are essential components

of the BER pathway. These results thus strongly support the

hypothesis that the decline of BER activity in brain

mitochondria may be an important contributory factor for

the age-dependent accumulation of endogenous oxidative

mtDNA damage.

Despite early studies showing that certain DNA damage,

such as pyrimidine dimers, was not repaired in mammalian

mitochondria (Clayton et al. 1974; LeDoux et al. 1993), a

number of later studies demonstrate that many forms of

oxidative DNA damage and chemical-induced base modifi-

cation or spontaneous base loss in mtDNA can be quickly

repaired (Driggers et al. 1993; LeDoux et al. 1993; Taffe

et al. 1996; Driggers et al. 1997; Anson et al. 1998; Grishko

et al. 1999). It has now been confirmed that the BER

pathway is the predominant mechanism responsible for the

repair of various oxidative DNA lesions in mammalian

mitochondria (for review see Croteau and Bohr 1997). BER

is a tightly controlled process that generally consists of four

steps (Dianov and Lindahl 1994): In the first step, oxidative

base damage or a modified base is removed by a specific

DNA glycosylase, resulting in an AP site; second, the DNA

sugar-phosphate backbone on the 5¢ side and the 3¢-terminalunsaturated aldehyde of an AP site are cleaved by AP

endonuclease, generating gaps in the strand; next, a DNA

polymerase fills in these gaps and resynthesizes one to five

new nucleotides; finally, the ends between the newly

synthesized nucleotides and the adjacent preexisting nucleo-

tides are sealed by a DNA ligase. With various in vitro

assays, the BER activity is readily detectable in mitochon-

dria of different organ tissues, including the liver, heart and

brain (Souza-Pinto et al. 1999; Chen et al. 2000). Further-

more, several repair enzymes involved in mitochondrial BER

have been partially purified and characterized, and showed

similar but not identical functional properties to those found

in the nucleus (Croteau et al. 1997; Rosenquist et al. 1997;

Lakshmipathy and Campbell 1999; Tell et al. 2001). It is

believed that the functional status of the BER pathway

largely determines the levels of endogenous oxidative

mtDNA damage (Kowald 2001). Strongly supportive of this

notion, a recent study demonstrated that the defection of the

OGG1 (oxoguanine DNA glycosylase)-initiated BER path-

way resulted in over 20-fold increases in the content of

8-oxodG in the mitochondrial genome (de Souza-Pinto et al.

2001).

The data presented here show that the brain mitochondrial

BER activity, measured based on the repair of 8-oxodG-

containing substrate, was markedly decreased in adult

(5-month-old) and aged (30-month-old) rats as compared

with prenatal or postnatal young rats. The decreased overall

BER activity in aged brains is clearly due to the decreased

activity of not only the 8-oxodG glycosylase, but also

enzymes responsible for each of the subsequent steps of

BER, including AP site incision, DNA polymerization and

DNA ligation. These age-dependent changes appear to be

unique to the central nervous system, as shown in recent

studies by de Souza-Pinto et al. which suggest that in liver

mitochondria, the 8-oxodG repair pathway is up-regulated

during aging (Souza-Pinto et al. 1999; de Souza-Pinto et al.

2001). This difference may explain why mitochondria in the

adult brain contain substantially higher baseline levels of

8-oxodG than that of the liver (Ames et al. 1993; Nagayama

et al. 2000; de Souza-Pinto et al. 2001; Hamilton et al.

2001a), and why there is an increased accumulation of

oxidative lesions in the aged brain compared with the young

adult brain (Mecocci et al. 1993; Hudson et al. 1998;

Hamilton et al. 2001b). Brain neurons are highly energetic

cells with a high rate of production of DNA-damaging ROS

in mitochondria (Floyd and Carney 1992; Chan 1996); it

appears that mtDNA accumulates higher levels of oxidative

lesions than nuclear DNA (Hudson et al. 1998; Barja and

Fig. 5 Age-dependent decline of DNA ligase activity in brain mito-

chondrial extracts. (a) Equal amount of protein (20 lg) from each age

was incubated with the 32P-labeled substrates at 30�C for 60 min,

generating mainly the 36-bp and, to the lesser extend, the 54-bp,

ligation product. Note that the amounts of the ligation products were

decreased in adult and aged rats. (b) Quantitative analysis of DNA

ligase activity in mitochondrial extracts as the function of age, deter-

mined by optical density measurements on autoradiographs from

three independent experiments. *p < 0.05 versus E17, P1w, P2w, or

P3w; #p < 0.05 versus E17, P1w, or P2w.

1280 D. Chen et al.


Herrero 2000; Hamilton et al. 2001b). The endogenous

antioxidant system, which constitutes the first line of defense

dedicated to reducing the levels of ROS, is insufficient to

completely prevent oxidative damage, even under physiolo-

gical conditions (Halliwell 1989; Murakami et al. 1998). Our

results further emphasize the importance of BER as the

second line of endogenous defense against oxidative damage

to the mitochondrial genome in brain.

Although all enzyme activities along the BER pathway

have been detected in mammalian mitochondrial extracts, the

identities of these enzymes are only partially elucidated thus

far. The role of OGG1 and DNA polymerase-c in mito-

chondrial BER has recently been confirmed (Longley et al.

1998; Chen et al. 2000; de Souza-Pinto et al. 2001). OGG1

is the main DNA glycosylase for the repair of 8-oxodG

lesions in DNA, and it contains an additional AP lyase

(b)

Fig. 6 Age-dependent changes in activities of DNA polymerase and

DNA ligase, detection using the reconstituted cell-free BER system.

(a) Schematic diagram illustrates the principle of the assay. The

50-mer oligonucleotide (partial sequence is shown) containing a uracil

at position 26 is annealed to the complementary oligonucleotide and

then treated with purified UDG (to generate an AP site) and endo-

nuclease IV (to generate a single-nucleotide nick). The addition of

purified DNA polymerase-b (b-pol) and T4 ligase to the reaction results

in the incorporation of [32P]GMP into the lesioned oligonucleotide.

(b) Representative autoradiograph shows the repair products from the

assay described in (a). Note that the addition of b-pol and T4 ligase

generated the 50-mer product, whereas the addition of b-pol alone

resulted in the 26-mer product only. (c) Mitochondrial extracts of

different ages (20 lg each) were incubated at 30�C for 60 min in the

presence of [a-32P]GTP with the DNA duplex that had been pretreated

with excess amounts of UDG and endonuclease IV. Note that both of

the 26-mer (representing the DNA polymerase activity alone) and

50-mer products (representing the combined activities of DNA

polymerase and DNA ligase) are decreased in the adult and aged rats.

(d) Quantitative analysis of DNA polymerase and ligase activities in

mitochondrial extracts as the function of age, determined by optical

density measurements of the 26- and 50-mer products on autoradio-

graphs from three independent experiments. *p < 0.05 versus E17,

P1w, P2w, or P3w.



activity that cleaves AP sites and produces a 3¢-deoxyribosemoiety and a 5¢-phosphate group via the a-eliminationmechanism (Bjoras et al. 1997; Radicella et al. 1997).

However, the brain mitochondria may also contain a yet-

to-be characterized APE which exhibits an APE class II

action, as suggested by our recent study which resolved the

3¢-end product generated by brain mitochondrial extracts inthe AP excision assay and identified a hydroxyl nucleotide

residue (Chen et al. 2000). Concerning the other key BER

enzymes, while the type of DNA ligase involved in

mitochondrial BER has not been determined, DNA poly-

merase-c is the only DNA polymerase found in mammalianmitochondria so far (Croteau and Bohr 1997). Accordingly,

in the present study, we measured the content of OGG1 and

DNA polymerase-c in mitochondrial extracts using westernblots in addition to the activity assays. The protein expres-

sion of both enzymes showed marked age-dependent down-

regulation, and the patterns of changes strikingly resembled

that of the activity assays. Thus, the decline of BER activity

during brain aging directly reflects the decreased content and

activity of BER enzymes in the mitochondria.

The mechanism responsible for the down-regulation of

mitochondrial BER enzymes in brain aging is not known.

Like most other mitochondrial metabolism enzymes, all

enzymes required for mitochondrial BER are transcription-

ally expressed in the nuclear genome and, upon translation,

imported into the mitochondria. The decreased contents of

these enzymes in the mitochondria may be due to the reduced

rate of gene expression at one or more of the above steps as

part of the general aging process. However, it is not the case

that all enzymes involved in mitochondrial metabolisms are

decreased in the brain as a function of age. For instance, the

levels of the major mitochondrial antioxidants such as

MnSOD and glutathione peroxidase were found to be either

increased or unchanged with age in the brain (Scarpa et al.

1987; Ceballos-Picot et al. 1992). Moreover, the levels of

cytochrome c oxidase IV, another nuclear genome-encoded

mitochondrial enzyme, were found to be increased in the

brain during aging (this study). The different patterns of gene

expression between mitochondrial BER enzymes and anti-

oxidants with age suggest that different mechanisms for gene

regulation may be involved. The age-dependent increases in

mitochondrial antioxidants have been attributed to the

endogenous adaptive responses to an increased production

of ROS in the aging process (Scarpa et al. 1987; Ames et al.

1993). Presumably, the machinery that controls the expres-

sion of BER enzymes may be retarded in response to the

slowly accumulated oxidative stress during aging. In

contrast, an acute oxidative stress may be a strong stimulus

up-regulating the expression of BER enzymes, as recent

studies demonstrate that a transient cerebral ischemic insult

can activate the expression of OGG1 and markedly boost

Fig. 7 Age-dependent regulation of expression of BER enzymes in

brain mitochondria. (a) Representative western blots show the

expression levels of Ogg1 and DNA polymerase-c in brain mitochon-

dria at different ages. Note that the levels of both BER enzymes were

decreased with age, whereas the levels of cytochrome c and cyto-

chrome c oxidase IV showed increases, and the levels of AIF

remained unchanged with age. (b) Shown are semiquantitative results

of relative abundance of the enzymes tested, as determined using

densitometry measurement on western blot autoradiographs. The

graphs illustrate mean ± SEM of arbitrary unit [region of optical density

(ROD) · area of band] measured on three individual western blots of

three different sets of brain samples, and all values were standardized

by that of E17. *p < 0.05 versus E17, P1w, or P2w; #p < 0.05 versus

E17, P1w, P2w, P3w, or P5m.

1282 D. Chen et al.


mitochondrial BER activity (Lin et al. 2000; Stetler et al.

2001). Further elucidation of the precise mechanism by

which the mitochondrial BER enzymes are regulated would

substantially enhance our understanding of the intrinsic

process of brain aging.

The findings from this study are significant. This study

provides direct evidence that the age-dependent decline in

BER activity occurs in brain mitochondria. This study has

also partially elucidated the mechanism responsible for the

decline of mitochondrial BER activity in aged brain. These

results thus point to a novel and potentially important

mechanism relevant to the brain aging process and mito-

chondrial dysfunction; the application of a means for

increasing mitochondrial BER capacity in the brain may be

a valuable strategy to minimize irreversible oxidative damage

to the mitochondrial genome during aging. Such an appli-

cation could have implications in the future preventive

management of various neurological disorders involving

mtDNA damage and mitochondrial dysfunction.

Acknowledgements

This work was supported by grants NS38560, NS36736 and

NS35965 (to JC) from NIH/NINDS. JC was also supported in part

by the Geriatric Research, Education and Clinical Center, Veterans

Affairs Pittsburgh Health Care System (Pittsburgh, PA, USA). We

thank Carol Culver for excellent editorial assistance and Pat

Strickler for secretarial support.

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