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* Laboratory of Molecular Gerontology, National Institute on Aging, National Institutes of Health, Baltimore, Maryland, USA; and
Gazi University, Faculty of Pharmacy, Toxicology Department, Hipodrom 06330 Ankara, Turkey
1Correspondence: 5600 Nathan Shock Dr., Box 1, Baltimore, MD 21224, USA. E-mail: bohrv{at}grc.nia.nih.gov
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: BER • repair activity • mouse tissues • oxidative stress • nucleus
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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180 guanines oxidize to 8-oxo-deoxyguanine (8-oxodG) per mammalian cell per day (1)
. Multiple mechanisms have evolved to repair DNA damage and preserve genome integrity. There are several different DNA repair pathways that include enzymes specialized for repair of a specific DNA lesion or type of DNA lesion (for a review, see ref 2
).
The molecular mechanisms of DNA repair have been studied extensively, but little is known about the physiological significance of different repair pathways in different tissues. Transgenic mice with reporter genes such as lacZ and lacI have been used to study and quantify spontaneous and induced mutagenesis in vivo. The results of these studies indicated that mutation frequency varies considerably with tissue (3)
and developmental stage (4)
. DNA damage accumulates at different rates in different tissues, especially in mitochondrial DNA (5)
; however, it is unclear whether these data reflect tissue-specific differences in DNA repair capacity or efficiency.
The base excision repair (BER) pathway repairs lesions involving modifications to the DNA bases, including lesions generated by reactive oxygen species. BER is initiated by a DNA glycosylase, which catalyzes the hydrolysis of the N-glycosyl bonds, releasing the base and generating an abasic site. The abasic site is cleaved by an AP lyase or AP endonuclease and, in most circumstances, the one base gap in the cleaved DNA strand is filled in by a DNA polymerase and ligated by a DNA ligase. The specificity of BER is provided by DNA glycosylases, which have precise substrate specificities. In mammalian cells there are four major DNA glycosylases: uracil DNA glycosylase (UDG), which removes uracil from DNA; oxoguanine DNA glycosylase (OGG1), which primarily recognizes 8-oxodG but is active on other oxidized purines; the human homologue of endonuclease III (NTH1), which recognizes and cleaves oxidized pyrimidines such as thymine glycol and 5-hydroxycitosine (5-OH-dC); and 3-methyl adenine DNA glycosylase, which removes alkylated bases (for a review, see ref 6
).
The initial observation that mitochondria are unable to remove UV-induced damage from their genome (7)
led to the notion that mitochondria are deficient in DNA repair capacity. However, this conclusion is not justified because mitochondria are proficient in BER (8)
. Mitochondria contain several BER enzymes, including DNA glycosylases, DNA ligase, and a deoxyribophosphate lyase activity associated with the mitochondrial polymerase 
(for a review, see ref 8
). It is not clear how the expression of mitochondrial isoforms of DNA repair enzymes is regulated and whether their expression is coordinated with expression of the nuclear DNA repair enzymes.
This study examines the tissue-specific distribution of BER activities in mitochondrial and nuclear extracts from C57/BL6 mice. UDG, OGG1, and NTH1 activities were measured in extracts from brain, liver, heart, testis, muscle, and kidney. Mitochondrial enzymes cytochrome c oxidase (COX) and citrate synthase (CS) were also measured to assess mitochondrial function. DNA repair activity was highest in nuclear and mitochondrial extracts from testis. Nuclear and the mitochondrial isoforms of each DNA glycosylase had similar levels of activity in a single tissue, suggesting that the two isoforms are coordinately regulated. In contrast, UDG1, OGG1, and NTH1 activities varied independently in different tissues, indicating these enzymes are not coordinately regulated.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Animals
We used male C57/BL6 mice at 6 months of age obtained from the National Institute on Aging animal colony. The animals were fed regular Purina animal chow ad libitum and kept in a 12 h light cycle. For mitochondrial isolation, the animals were killed by cervical dislocation and tissues were immediately removed and processed. All experiments were performed in accordance with the Guidelines for the Use and Care of Laboratory Animals (NIH Publication 85–23).
Oligonucleotides
The 8-oxodG, uracil, and control oligonucleotides were from Midland Certified Reagent Co. (Midland, TX) The 5-OH-dC oligonucleotide was kindly provided by Dr. Michelle Ham (Massachusetts Institute of Technology, MIT, Cambridge, MA). All oligonucleotide sequences are presented in Table 1
. Oligonucleotides containing either the DNA lesion or an unmodified base were 5'-end-labeled using T4 polynucleotide kinase and [-32P] ATP. To separate the unincorporated free [-32P] ATP, reaction mixtures were spun through a G25 column. Complementary oligonucleotides were annealed in 10 mM Tris, 1 mM EDTA, 100 mM KCl by heating the samples to 80°C for 5 min and allowing them to cool slowly to room temperature.
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Preparation of mitochondria
Mitochondria were isolated from the different mouse tissues using standard procedures from the literature that have been optimized for the particular characteristics of each tissue yield intact and functional mitochondria. Because each tissue has unique structural characteristics, different steps may be required for efficient release of mitochondria from the intracellular environment. In heart and muscle, the release of mitochondria depends on the disruption of the myofibril net with a protease treatment (9
, 10)
. In brain, where most mitochondria localize to the axonal arm, the disruption of the tissue with homogenization causes the mitochondria to be encapsulated in plasma membrane vesicles, termed synaptosomes. To disrupt these and release free mitochondria, a mild detergent treatment is required (11)
. In soft tissues such as liver, kidney, and testis, most mitochondria are released after homogenization; thus, only the differential centrifugations and density gradients are required to obtain good yield of intact mitochondria (12)
. All procedures were carried out on ice or at 4°C. The solutions were sterilized using 22 µm Millipore filters. The final mitochondrial pellets were stored at -80°C until use. Protein concentrations were determined using the Lowry method, with BSA as standard.
Isolation of kidney and liver mitochondria
Mouse kidney and liver mitochondria were isolated using a combination of differential centrifugation and Percoll gradient, as described (13)
, using MSHE (0.21 M mannitol, 70 mM sucrose, 10 mM HEPES (pH 7.4), 1 mM EGTA, 2 mM EDTA, 0.15 mM spermine, and 0.75 mM spermidine), with the following protease inhibitors added just before use: 5 mM DTT, 2 µ g/mL leupeptin, 2 µM benzamide-HCl.
Isolation of brain mitochondria
The brains were removed and washed free of blood with MSHE buffer. The chopped tissue was then transferred to a glass/glass homogenizer and homogenized with 15 strokes. Suspensions were then incubated on ice for 30 min to promote cellular lyses. Homogenates were centrifuged at 1200 g for 12 min and the pellets were stored for nuclear extract preparation. Supernatants were centrifuged at 10,000 g for 15 min. The pellets were suspended in 3 mL of 3% Ficoll 400 (in 1/2xMSHE) and gently layered onto 3 mL of 6% Ficoll in 15 mL tubes. The gradients were centrifuged at 10,400 g for 25 min. Mitochondrial pellets were suspended in 300 µL 1xMSHE and incubated for 15 min on ice in the presence of 0.1% digitonin. Mitochondrial suspensions were centrifuged at 10,400 g for 15 min and pellets were washed in 1 mL of 1xSHE.
Isolation of heart and muscle mitochondria
Heart and muscle mitochondria were isolated using a combination of high-speed differential centrifugation and a short protease digestion with Nagarse, as described previously (9)
, with the modification that the isolation buffer was substituted with MSHE.
Isolation of testis mitochondria
The testes were homogenized in MSHE and the crude homogenate was centrifuged at 500 g for 15 min. Pellets were stored for nuclear extracts and supernatants were then centrifuged at 10,000 g for 10 min. To ensure that the mitochondrial fractions were not contaminated with cellular debris, the pellet was suspended in MSHE and centrifuged again at 500 g, 10 min. The supernatant was then centrifuged at 10,000 g for 15 min to yield the final mitochondrial pellet.
Preparation of nuclear extracts
Nuclear extracts were prepared from the pellets obtained after the first low-speed spin, during the differential centrifugations for the isolation of mitochondria as described (14)
. The extracts were divided and stored at -80°C until use.
Measurement of DNA repair activities
Different DNA glycosylase activities were measured using an oligonucleotide incision assay with single-lesion DNA substrates (Table 1)
. All reactions were carried out as described before (15)
. The amount of extract added to each reaction varied according to the experiment as specified in the figure legends. All reactions were incubated at 37°C for 6 h (8-oxo-dG and 5-OH-dC) or for 1 h (uracil).
Measurement of citrate synthase and cytochrome c oxidase activity
CS activity was measured spectrophotometrically (9)
. COX activity was measured as the oxygen consumption rate, using a Clark type oxygen electrode in the presence of oxidized cytochrome c and with tetramethyl-p-phenyldiamine (TMPD)/ascorbate as the electron donor.
Western analysis
Mitochondrial protein (20–50 µg) was separated on12% Tris-glycine gels (Novex). Transfer to PVDF membranes (Novex) was done by electroblotting in transfer buffer (12 mM Tris, pH 8.3; 96 mM glycine, 20% methanol) for 2 h at 30 V. The membrane was blocked for 16 h at 4°C in 5% non-fat dry milk (Bio-Rad, Hercules, CA) in TBST (20 mM Tris-HCl pH 7.2, 137 mM NaCl, 0.1% Tween-20). Fresh milk-TBST was added with the primary antibody, which was either mouse monoclonal anti-lamin B2 (Novocastra) or mouse monoclonal anti-cytochrome oxidase IV (Molecular Probes, Eugene, OR). Detection was performed with ECL+Plus® (Amersham-Pharmacia Biotech, Piscataway, NJ).
Statistical analysis
The results presented are an average of six different mitochondrial and nuclear preparations for each tissue. Each sample was assayed twice in duplicate. The differences among tissues were analyzed by the Student’s t test and a P < 0.05 was considered statistically significant.
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RESULTS |
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shows a typical Western blot assessing the quality of mitochondrial extracts from mouse brain, liver, heart, muscle, testis, and kidney. Nuclear extracts from liver were included as a positive control. The blot was hybridized with anti-cytochrome oxidase IV (COX) and anti-lamin B2. Only marginal amounts of lamin B 2 were detected in mitochondrial extracts from muscle, heart, liver, kidney, and brain (Fig. 1
, upper panel). The intensity of the lamin B2 band corresponded to < 8% of the mitochondrial content in those five tissues, as assessed by the intensity of the COX IV band (Fig. 1
, lower panel). More significant amounts of lamin B2 (20.1% of the COX VI signal) were detected in testis mitochondrial extracts. To determine whether nuclear contamination could account for the DNA glycosylase activities measured here, we treated testis mitochondria with proteinase K to remove enzymatic activity not contained inside the mitochondria. OGG1 incision activity did not decrease after proteinase K treatment (data not shown), indicating that nuclear DNA repair proteins do not contribute to the DNA repair activity detected in mitochondrial extracts.
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UDG, OGG1, and NTH1 activities were measured in nuclear and mitochondrial extracts using an incision assay and DNA oligonucleotide substrates containing one DNA lesion per oligonucleotide (oligonucleotide substrates are shown in Table 1
). Initially, the amount of protein extract was varied to determine the linear range of the assay for each extract and oligonucleotide (data not shown). All subsequent assays were carried out in the linear range of the incision assay.
Figure 2
shows typical autoradiograms of the reactions for OGG1, NTH1, and UDG1 using oligonucleotide DNA substrates containing 8-oxodG (Fig. 2A, D
), 5-OH-dC (Fig. 2B, E
), and uracil (Fig. 2C, F
). Reaction products were separated on a denaturing polyacrylamide gel. A negative control reaction was carried out with a lesion-free DNA oligonucleotide. For the 8-oxodG-containing DNA substrate, the bacterial enzyme Fpg was used as a positive control. For the 5-OH-dC oligonucleotide we used the bacterial enzyme Endo III and for the uracil-containing substrate we used recombinant UDG, followed by pyridine treatment (data not shown). After removal of the damaged base by the DNA glycosylase, the abasic site containing substrate was cleaved by either the associated AP lyase activity or an alternate AP endonuclease, generating the incision product (P), which is visualized as a faster migrating species. We observed that two slightly different sized products were generated upon incubation of all three oligonucleotide substrates with the extracts. This probably reflects the processing of the abasic site by more than one distinct endonuclease/lyase activity, generating products with different ends.
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Nuclear and mitochondrial OGG1 and NTH1 activities were quantified and compared in extracts from mouse tissues (Fig. 3
A, B). OGG1 activity varied significantly between tissues whereas there was a concordance between the relative tissue-specific levels of incision between nuclear and mitochondrial extracts. The highest OGG1 activity was in nuclear and mitochondrial extracts from testis. OGG1 activity was similar in mitochondrial extracts from liver and kidney but not for nuclear extracts from liver and kidney. OGG1 activity was lower in mitochondrial and nuclear extracts from muscle, heart, and brain than in liver, kidney, and testis (Fig. 3A
). NTH1 activity was relatively uniform in extracts from different mouse tissues (Fig. 3B
). Mitochondrial NTH1 was higher in heart and testis, but nuclear NTH1 was lower in heart than in other tissues. In most tissues, nuclear NTH1 activity was higher than that of mitochondrial NTH1, and there was less concordance between nuclear and mitochondrial activities of NTH1 than observed for OGG1. OGG1, and NTH1 (Fig. 3A, B
) activities are assessed at the same scale; it appears that NTH1 activity was higher than OGG1 activity in most tissues. These results indicate that OGG1 activity is more coordinately regulated than NTH1 activity.
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UDG activity varied in nuclear and mitochondrial extracts from different tissues (Fig. 4
). Relative expression levels of UDG in the different tissues was similar for the nuclear and the mitochondrial extracts aside from heart, suggesting that the mitochondrial isoform of UDG might be regulated coordinately with the nuclear UDG.
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To directly compare the relative levels of the three DNA glycosylases in all tissues investigated, Fig. 5
shows the relative levels of UDG, OGG1, and NTH1 activity in mitochondrial extracts from the six different tissues. In general, UDG activity was higher than the activity of the other two glycosylases. In muscle, testis, brain, and heart, UDG activity was severalfold higher than OGG1 and NTH1 activities. We observed that in most tissues the relative activity of these three DNA glycosylases follows the same pattern, with UDG having the highest activity, followed by NTH1 and OGG1.
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To account for possible variation in mitochondrial content between different tissues, DNA repair activity was normalized to CS. CS is widely used as a mitochondrial marker because CS activity (9)
and mRNA (16)
are constitutively expressed and do not change with age or pathological condition. Normalizing OGG1, NTH1, and UDG activity to CS did not alter the relative results (Table 2
).
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DNA repair efficiency was compared in the nuclear and mitochondrial compartment of each tissue. Enzyme-specific activity was measured in nuclear and mitochondrial extracts and the ratio of mitochondrial:nuclear incision activity was calculated. The results are presented in Table 3
. In most tissues, enzyme-specific activity was 50% lower in the mitochondrial extract than in the nuclear extracts. However, this pattern was not observed in extracts from heart, where UDG, NTH1 and OGG1 enzyme-specific activities are twofold higher in the mitochondrial extract than in the nuclear extract (Table 3)
.
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To investigate a possible relationship between mitochondrial function and DNA repair activity in these organelles, COX activity was measured in mouse mitochondrial extracts by following oxygen consumption in the presence of cytochrome c and TMPD/ascorbate as an electron donor. COX activity varied in the mouse extracts (data not shown): heart and brain had high COX activity; liver had intermediate COX activity; and testis, muscle, and kidney had low COX activity (data not shown). However, no obvious relationship between COX activity and DNA repair activities was observed.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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demonstrated that human OGG1 undergoes phosphorylation in vivo. However, this modification did not cause any significant change in hOGG1 catalytic activity (17)
. Thus, the BER repair capacity for several common DNA lesions (8-oxodG, 5-OH-dC and uracil) varies in a tissue-specific manner.
Oxidative stress and oxidative DNA damage varies considerably in different tissues and especially in mitochondria. Hamilton et al. (18)
recently showed that the level of 8-oxodG in mitochondrial DNA from liver, heart, and brain was 6-, 16-, and 23-fold higher than in nuclear DNA, respectively. This result suggests that tissues with higher oxidative load accumulate more oxidative DNA damage in mitochondrial DNA than in nuclear DNA.
Testis has higher nuclear and mitochondrial DNA glycosylase activity than all other tissues (i.e., brain, liver, heart, muscle, and kidney) examined in this study. This is consistent with previous studies demonstrating high repair capacity in testis nuclei and the observation that many DNA repair genes are highly expressed in testis (19
20
21)
. This may indicate that BER and other DNA repair pathways are essential for maintaining genomic integrity during gametogenesis. In Saccharomyces cerevisiae, mRNA levels of DNA repair genes such as RAD18, RAD2, RAD6, RAD7, and RAD23 are elevated during meiosis but remain unchanged during the mitotic cell cycle (22)
. This supports the essential role of DNA repair during gametogenesis. Although sperm cells do not contribute a significant amount of mitochondrial DNA to the embryo, the integrity of the mitochondrial genome and mitochondrial function is likely to be important to proper sperm function. For example, sperm without functional mitochondria may be at a selective disadvantage because of an inadequate supply of ATP for flagellar movement and mobility.
It is widely accepted that postmitotic cells and tissues have decreased DNA repair capacity. Experimental support for this idea comes from data showing that unscheduled DNA synthesis (UDS) decreases during differentiation of a myogenic cell line (23)
. UDS decreases in rat urogenital epithelial cells exposed to N-nitrosomorpholine while UDS increases significantly in the liver (24)
. In addition, expression of the human mutY homologue decreases in postmitotic tissues (25)
. However, results presented in this study indicate that postmitotic tissues such as heart and brain express DNA glycosylases at a similar or higher level than liver. Moreover, OGG1 activity in rat heart increases significantly with age (15)
. Li et al., 2001 (26)
demonstrated that heart failure induced by TNF-
was associated with impaired DNA repair activity, pointing to the role of DNA repair in maintaining cardiac function. Kanoh et al. (27)
found significant DNA in situ nick end-labeling in heart tissue from patients with dilated cardiomyopathy, and they speculated that this reflected DNA repair capacity. In brain, up-regulation of BER activity was detected in the forebrain region of mouse brain after ischemia-reperfusion injury (28)
, and the expression of APendonuclease was shown to increase significantly in hippocampus after global ischemia (29)
. Several DNA repair deficient knockout mice have neurological abnormalities or severe postnatal neurodegeneration, suggesting that DNA repair mechanisms are very important in the brain (30)
. These results suggest that DNA repair is active and plays an important role in brain and heart.
This study investigates the level of expression of mitochondrial and nuclear DNA glycosylase activities for several DNA base lesions in mouse tissues. Nuclear and mitochondrial isoforms of OGG1, NTH1, and UDG were expressed at similar activity levels in kidney, muscle, liver, testis, and brain, but were not similar in heart. Mitochondrial NTH1 and UDG activities, but not that of OGG1, were higher than their nuclear isoforms. These enzyme isoforms are encoded by a single nuclear gene (25
, 31)
. Thus, the expression of both isoforms is somewhat coordinated. On the other hand, their expression is different in different mouse tissues. The expression pattern of both isoforms of the murine UDG gene (ung1, the mitochondrial isoform, and ung2, the nuclear) has been investigated by Nilsen et al. (32)
. Results reported by those authors are highly consistent with the activity levels reported here. We observed mitochondrial UDG activity to be higher in tissues with high mitochondrial content, such as heart and muscle.
In conclusion, our results show that BER capacity varies greatly among different tissues in the mouse. The mitochondrial and nuclear BER glycosylases in each organ correlate well. Nuclear activities strongly correlate with the proliferative status of the tissue, and this points to a role for BER during DNA replication. On the other hand, the mitochondrial isoforms were higher in tissues with high-energy demand, such as heart and muscle, suggesting that DNA repair may play a very significant role in maintaining mitochondrial integrity. The mitochondrial OGG1 activity, however, was relatively lower in heart and muscle as well as in brain. Because heart and brain are postmitotic tissues, it has been postulated that accumulation of DNA damage in these tissues is a critical determinant for life span (33)
. Higher levels of 8-oxodG were detected in rat brains than other tissues such as kidney and lung (34)
, and these have been shown to increase significantly with age in heart and brain (18)
. Thus, our observation that these tissues have relatively lower OGG1 activity, and therefore remove 8-oxodG more slowly, may explain the age-associated accumulation of oxidative damage, which may be the cause of age-associated loss of function.
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ACKNOWLEDGMENTS |
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Received for publication May 16, 2002.
Revision received August 23, 2002.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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