Mitochondrial transcription factor A overexpression and
base excision repair deficiency in the inner ear of rats with
D-galactose-induced aging
Yi Zhong
1,
*, Yu-Juan Hu
1,
*, Bei Chen
1,2
, Wei Peng
1
, Yu Sun
1
, Yang Yang
1
, Xue-Yan Zhao
1
,
Guo-run Fan
1
, Xiang Huang
3
and Wei-Jia Kong
1
1 Department of Otorhinolaryngology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan,
China
2 Department of Otorhinolaryngology, The First Affiliated Hospital of Zhengzhou University, China
3 Institute of Otorhinolaryngology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
Introduction
Age-related hearing loss, also known as presbycusis, is
a universal feature of mammalian aging, and is the

and frequent form of mtDNA damage associated with aging and degenera-
tive diseases. The accumulation of the mitochondrial common deletion has
been proposed to play a crucial role in age-related hearing loss (presbycu-
sis). However, the mechanisms underlying the formation and accumulation
of mtDNA deletions are still obscure. In the present study, a rat mimetic
aging model induced by
D-Gal was used to explore the origin of deletion
mutations and how mtDNA repair systems modulate this process in the
inner ear during aging. We found that the mitochondrial common deletion
was greatly increased and mitochondrial base excision repair capacity was
significantly reduced in the inner ear in
D-Gal-treated rats as compared
with controls. The overexpression of mitochondrial transcription factor A
induced by
D-Gal significantly stimulated mtDNA replication, resulting in
an increase in mtDNA copy number. In addition, an age-related loss of
auditory sensory cells in the inner ear was observed in
D-Gal-treated rats.
Taken together, our data suggest that mitochondrial base excision repair
capacity deficiency and an increase in mtDNA replication resulting from
mitochondrial transcription factor A overexpression may contribute to the
accumulation of mtDNA deletions in the inner ear during aging. This
study also provides new insights into the development of presbycusis.
Abbreviations
BER, base excision repair; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; OGG1, 8-oxoguanine glycosylase; OHC, outer hair cell;
OS, oxidative stress; Pol-c, DNA polymerase-c; ROS, reactive oxygen species; TFAM, mitochondrial transcription factor A; VDAC, voltage-
dependent anion channel.
2500 FEBS Journal 278 (2011) 2500–2510 ª 2011 The Authors Journal compilation ª 2011 FEBS
by cell death, when the proportion of mutant mtDNA
exceeds a certain threshold level [4]. However, the

tem of rats with d-Gal-induced aging [11]. As is well
known, besides the degeneration of the central audi-
tory system, the age-related changes of the peripheral
auditory system also play an important role in the
development of presbycusis. However, as the organ of
Corti (part of the peripheral auditory system) is tiny
and extremely difficult to dissect, investigations in the
peripheral auditory system are much more difficult
than those in the central auditory system. Up to now,
the effect of BER on large-scale mtDNA deletions in
the peripheral auditory system of rats with d-Gal-
induced aging is still unknown.
Mitochondrial transcription factor A (TFAM) plays
important roles in mtDNA replication and transcrip-
tion, and the structure ⁄ organization of mitochondrial
nucleoids [12–14]. Moreover, TFAM has been previ-
ously reported to modulate BER in mitochondria by
virtue of its DNA-binding activity and protein interac-
tions [15]. Additionally, relaxed replication of mtDNA
within single cells was suggested to be associated with
the clonal expansion of single mutant events during
human life [16]. Thus, TFAM may be implicated in
mutation events. However, a function for TFAM in
mtDNA deletion formation has not yet been investi-
gated.
We have previously utilized overdoses of d-Gal to
induce OS in vivo, to mimic natural aging of rats [17].
The rats with accelerated aging induced by d-Gal may
harbor the mtDNA 4834-bp deletion (also known as
the common deletion) in the inner ear as well in as

was specific for the new fusion sequence, which was
present only in mutant mtDNA harboring the com-
mon deletion. By applying the specific probe, we can
even detect the presence of the common deletion in the
inner ear in the control group. The proportions of the
common deletion in the inner ear in the low-dose,
medium-dose and high-dose groups of d-Gal-treated
rats were 9.60% ± 1.46%, 11.31% ± 1.64%, and
16.03% ± 2.30%, respectively, which were signifi-
cantly greater than those of the control group
(4.35% ± 0.46%) (P < 0.05) (Fig. 1). Moreover,
the deletion burden in the high-dose d-Gal group was
Y. Zhong et al. Accumulation of mtDNA deletions in inner ear
FEBS Journal 278 (2011) 2500–2510 ª 2011 The Authors Journal compilation ª 2011 FEBS 2501
significantly higher than that in the low-dose d-Gal
group (P < 0.05), but there was no difference in the
deletion burden between the low-dose and medium-
dose groups (P > 0.05).
Mitochondrial DNA proliferation induced by
D-Gal
To investigate the effect of d-Gal on mtDNA prolifer-
ation, we quantified the relative abundance between
the mitochondrial D-loop region and a nuclear gene
(b-actin) by quantitative PCR assay. As shown in
Fig. 2, the relative mtDNA copy numbers in the inner
ear were increased by 2.1-fold, 2.5-fold and 3.8-fold in
the low-dose, medium-dose and high-dose groups,
respectively, as compared with those in controls
(P < 0.05). No significant difference was found
between the low-dose and medium-dose groups

time PCR experiments for the essential BER enzymes,
Pol-c and OGG1, were performed. As shown in Fig. 5,
mRNA levels of both Pol-c and OGG1 were signifi-
cantly reduced in the d-Gal groups as compared with the
Fig. 2. Effect of D-Gal on the amount of total mtDNA in the inner
ear. Relative mtDNA copy numbers were significantly increased in
D-Gal-treated groups as compared with the control group.
*P < 0.05, **P < 0.01 versus control group, n = 6. LD, low-dose
D-Gal group; MD, medium-dose D-Gal group; HD, high-dose D-Gal
group.
Fig. 3. Quantitative analysis of TFAM mRNA expression in the
inner ear of experimental groups. The expression levels of TFAM
were significantly increased in
D-Gal-treated rats as compared with
control rats. *P < 0.05, ** P < 0.01 versus control group, n = 6. LD,
low-dose
D-Gal group; MD, medium-dose D-Gal group; HD, high-
dose
D-Gal group.
Fig. 1. Effect of D-Gal on the amount of the mitochondrial common
deletion in the inner ear. The percentages of the mitochondrial
common deletion in
D-Gal-treated groups were significantly higher
than in the control group. *P < 0.05, **P < 0.01 versus control
group, n = 6. LD, low-dose
D-Gal group; MD, medium-dose D-Gal
group; HD, high-dose
D-Gal group.
Accumulation of mtDNA deletions in inner ear Y. Zhong et al.
2502 FEBS Journal 278 (2011) 2500–2510 ª 2011 The Authors Journal compilation ª 2011 FEBS

organ of Corti from the high-dose d-Gal group. They
were taken from the basal turn, close to the hook
region, of a cochlea from one high-dose d-Gal sub-
ject. Small amounts of OHC loss are evident in this
image.
Fig. 5. Quantitative analysis of Pol-c and OGG1 mRNA expression
in different groups. The expression levels of Pol-c and OGG1 were
significantly decreased in
D-Gal-treated groups as compared with
the control group. **P < 0.01 versus control group, n = 6. LD, low-
dose
D-Gal group; MD, medium-dose D-Gal group; HD, high-dose
D-Gal group.
Fig. 4. Western blotting and densitometry analysis of TFAM pro-
tein expression in the inner ear. (A) Representative western blots
show the expression levels of TFAM in different groups. (B) The
relative abundance of TFAM protein was significantly increased in
the inner ear of
D-Gal-treated rats as compared with controls.
**P < 0.01 versus control group, n = 12. LD, low-dose
D-Gal group;
MD, medium-dose
D-Gal group; HD, high-dose D-Gal group.
Fig. 6. Western blotting and densitometry analysis of Pol-c and
OGG1 protein expression in the inner ear. (A) Representative wes-
tern blots show the expression levels of TFAM in different groups.
(B) The relative abundance of Pol-c and OGG1 protein was signifi-
cantly decreased in the inner ear of
D-Gal-treated rats as compared
with controls. **P < 0.01 versus control group, n = 12. LD, low-

of presbycusis.
In the present work, we demonstrated significantly
increased levels of the mitochondrial common deletion
in the inner ear of rats with d-Gal-induced aging.
Mitochondrial DNA deletions can accumulate with
aging in postmitotic tissues with high energetic
demands, such as skeletal muscle, heart, brain, and the
inner ear. Therefore, mtDNA deletions have been con-
sidered to represent an important molecular marker
during aging. Among numerous deletions, the mito-
chondrial common deletion (4977 bp and 4834 bp in
humans and rats, respectively) is the most typical and
frequent form of mtDNA damage associated with
aging [23–25]. A recent investigation demonstrated a
significant association between the level of the mito-
chondrial common deletion in human cochlear tissue
and the severity of hearing loss in individuals with
presbycusis [26]. Accumulation of the mitochondrial
common deletion was proposed to play a critical role
in the development of presbycusis. During the aging
process, both deleted and wild-type mtDNA can coex-
ist in a state called heteroplasmy, but the ratio of
mutated to wild-type mtDNA may vary widely
between different tissues, and even between cells within
A
B
C
D
Fig. 7. A representive image showing mild
OHC loss in the organ of Corti from the

mitochondria, and consists of two subunits: a catalytic
subunit, with both polymerase and proofreading 3¢–5¢
exonuclease activity, and an accessory subunit, which
confers processivity. A proofreading-deficient version
of Pol-c is associated with the accumulation of
mtDNA deletions, and premature onset of aging-
related phenotypes in knock-in mice [27]. In addition,
OGG1 also has a crucial role in the repair of oxidative
damage in mammalian mitochondria [28], and it is the
primary enzyme for the repair of 8-oxo-deoxyguanine
lesions, which constitute one of the major base modifi-
cations following oxidative damage to mtDNA [29].
8-Oxo-deoxyguanine is strongly mutagenic, having the
propensity to mispair with adenines, leading to
increased frequency of a spontaneous G–C to T–A
transversion, which, in turn is related to tumors, aging,
and degenerative disease [30]. Using the technique of
ligation-mediated PCR, Driggers et al. [31] found that
the mitochondrial common deletion could be initiated
by persistent oxidative damage in rat mtDNA at a sin-
gle guanine at one of the break sites. In OGG1-knock-
out mice, the level of 8-oxo-deoxyguanine in the
mtDNA was significantly higher than in wild-type mice
[28]. In our study, increased mtDNA deletion load and
decreased levels of mtDNA repair enzymes (Pol-c and
OGG1) were observed in the inner ear of rats with
d-Gal-induced aging. The data suggested that the
downregulation of mitochondrial BER expression may
be an important contributor to increased oxidative
mtDNA damage in the inner ear during aging. A pre-

within individual cells [16,36]. The replication of
mtDNA with deletion mutations does not occur at the
expense of wild-type mtDNA replication, but deleted
mtDNA molecules are advantaged [37]. Additionally,
treatments that enhance mtDNA replication, such as
vigorous excercise, could also amplify the process of
the clonal expansion of mtDNA mutations, with
potentially detrimental long-term consequences [38].
However, in postmitotic cells such as neurons, there is
no cell division, and mtDNA replication frequency is
fairly low in these cells. In our study, we found a sig-
nificant increase in mtDNA copy number in the inner
ear of rats with d-Gal-induced aging. This finding sug-
gested that replication of mtDNA is relatively active in
the inner ear exposed to d-Gal, although the inner ear
is a postmitotic tissue. In addition to mitochondrial
BER deficiency, the relatively active mtDNA replica-
tion induced by d-Gal exposure, which probably pro-
vides more opportunities for replication errors and
clonal expansion of mutation events, may partially
explain the increased mutation load in the inner ear.
In our study, increases in TFAM expression and dele-
tion mutation load were demonstrated in the inner ear of
d-Gal-treated rats. The replication of mtDNA is con-
trolled by nuclear-encoded transcription and replication
factors that are translocated to the mitochondria. Both
TFAM and mitochondrial-specific Pol-c are required.
TFAM is a key factor involved in directly regulating
Y. Zhong et al. Accumulation of mtDNA deletions in inner ear
FEBS Journal 278 (2011) 2500–2510 ª 2011 The Authors Journal compilation ª 2011 FEBS 2505

genesis was associated with oxidative stress induced by
d-Gal. Increased mitochondrial ROS production has
also been reported to be associated with increased muta-
tion load and mitochondrial biogenesis in other patho-
logical settings [44,45], similar to the age-related one
induced by d-Gal treatment in the present study. Such
an OS condition may induce increased TFAM levels and
increased mtDNA replication as a compensatory
response for the decreased mitochondrial functionality.
However, it is likely that TFAM overexpression induced
by d-Gal is a double-edged sword. TFAM overexpres-
sion, as a defense response to dysfunctional oxidative
phosphorylation during aging, results in compensatory
amplification of mtDNA, which may rescue age-depen-
dent impairment in mitochondrial functions [46]; mean-
while, increased mtDNA replication may involve clonal
expansion of mtDNA deletions and abnormal nucleoid
enlargement, which could amplify the effect of mito-
chondrial BER deficiency on mtDNA mutations. Taking
into account mitochondrial BER deficiency (especially
Pol-c defeciency) in the inner ear induced by d-Gal,
increased mtDNA replication may be a potential con-
tributor to the accelerated accumulation of mtDNA
mutation load, and ultimately severe respiratory chain
deficiency, in individual cells, followed by cell death.
In conclusion, this investigation demonstrates that a
significant decline in mitochondrial BER expression and
a remarkable increase in mtDNA replication resulting
from TFAM overexpression are involved in the accumu-
lation of mtDNA deletion mutations. Enhanced muta-

DNA extraction
After the last injection, 24 animals (n = 6 from each
group) were killed, and bilateral cochleae from each rat
were rapidly removed. The membranous labyrinth tissues
were then harvested from cochleae with an anatomy micro-
scope. Samples were stored at ) 80 °C until processing.
One side of the cochleae was used for mtDNA analysis,
and the other was used for RNA extraction (see below).
Total DNA was extracted with the standard SDS–protein-
ase K method. The DNA concentration of each sample was
assayed with the gene quant pro dna ⁄ rna calculator
(BioChrom, Cambridge, UK).
Quantification of the mitochondrial common
deletion
The proportion of the mitochondrial common deletion was
determined with a TaqMan real-time PCR assay. The
Accumulation of mtDNA deletions in inner ear Y. Zhong et al.
2506 FEBS Journal 278 (2011) 2500–2510 ª 2011 The Authors Journal compilation ª 2011 FEBS
mitochondrial D-loop region is rarely deleted, and the copy
number of this region therefore serves as a measure of the
total amount of mtDNA in a given tissue sample. The Taq-
Man PCR assay primers and probes for mitochondrial D-
loop region and common deletion were previously described
by Nicklas et al. [24]. The quantitative real-time PCR assay
was performed on the ABI Prism 7900HT Fast Real-Time
PCR System (Applied Biosystems, Foster City, CA, USA)
in a 20-lL reaction mixture containing 4.6 lL of distilled
water, 4 lL of sample DNA ( 40 ng of DNA), 10 lLof
2· TaqMan PCR mix (Takara, Dalian, China), 0.4 lLof
50· ROX reference dye, 0.4 lLof10lm each forward and

cycling conditions were as follows: one cycle of 30 s
at 95 °C, and 40 cycles of 95 °C for 5 s and 60 °C for
30 s. The mtDNA copy number was calculated from DCt
(Ct
D-loop
) Ct
b-actin
), where the mean amount of mtDNA
per cell = 2 (2
)DCt
), to account for the two copies of the
b-actin gene in each cell nucleus. The mtDNA copy number
of the control group was taken as the reference point to
calculate the relative mtDNA copy number of the experi-
mental group.
RNA preparation and quantitative real-time PCR
The mRNA expression levels of TFAM, Pol-c and OGG1
were determined by quantitative real-time PCR. Total RNA
of membranous labyrinth tissues was extracted with Trizol
reagent (Invitrogen, Carlsbad, CA, USA), according to the
manufacturer’s protocol. cDNA synthesis was performed
with 1 lg of total RNA, with a ReverTra Ace reverse trans-
criptase kit (Toyobo Co. Ltd. Osaka, Japan). The RNA and
cDNA of each sample were assayed with the gene quant
pro dna ⁄ rna calculator to assess concentrations and
purification. cDNA samples were stored at ) 20 °C until
use. Quantitative real-time PCR was performed by applying
the real-time SYBR Green PCR technology with the use of a
BioRad Opticon 2 genetic analyzer (Bio-Rad Laboratories,
Hercules, CA, USA). Validated primers for each gene were

Tissue Mitochondria Isolation Kit (Beyotime Institute of
Biotechnology, China), according to the manufacturer’s
instructions. Protein concentrations were determined with
the BCA Protein Assay Kit (Pierce Biotech, Rockford, IL,
USA), with BSA as standard.
Western blot analysis
Mitochondrial protein ( 30 lg) was loaded on 15% or
6% SDS ⁄ PAGE gels, and transferred to a poly(vinylidene
difluoride) membrane. After protein transfer, the mem-
branes were blocked with 5% nonfat milk in NaCl ⁄ Tris
and incubated with goat polyclonal antibody against
TFAM (1 : 500; Santa Cruz Biotechnology, Santa Cruz,
CA, USA), OGG1 (1 : 1000; Abcam, Cambridge, MA,
USA), Pol-c (1 : 500; Santa Cruz Biotechnology), and
Y. Zhong et al. Accumulation of mtDNA deletions in inner ear
FEBS Journal 278 (2011) 2500–2510 ª 2011 The Authors Journal compilation ª 2011 FEBS 2507
voltage-dependent anion channel (VDAC) (1 : 1000;
Abcam, Cambridge, MA, USA). VDAC, a specific mito-
chondrial membrane protein, was used as a loading control
for mitochondrial protein. Secondary anti-goat and anti-
rabbit IgG (Santa Cruz Biotechnology) was applied at a
dilution of 1 : 3000–1 : 5000. Membranes were then
washed, and proteins were visualized with ECL plus (Pierce
Biotech). The blots were scanned, and relative band density
was analyzed with the gel-pro application (Media Cyber-
netics, Silver Spring, MD, USA). The densities were
normalized to VDAC.
Morphological analysis of the cochlea
After decapitation, cochleae from 16 animals (n = 4 from
each group) were removed immediately from the temporal

i
, the sections of
the organ of Corti (base to apex), corresponding to apical,
middle and basal portions of the cochlea, were mounted on a
slide with antifade mounting media, and imaged with a laser
scanning confocal microscope (Olympus, Tokyo, Japan).
Statistical analysis
Data are presented as mean ± standard error of the mean.
Analysis was performed with spss 13.0 software (IBM,
Armonk, NY, USA). Statistical significance was tested with
one-way ANOVA. The least significant difference post hoc
test was used to evaluate the differences between two of the
groups. Differences with a P-value < 0.05 were considered
to be statistically significant.
Acknowledgements
This work was supported by grants from the National
Nature Science Foundation of China (Nos. 30730094,
30872865, and 81000409), the National High Technol-
ogy Research and Development Program of China
(863 Program) (No. 2008AA02Z428), the Major State
Basic Research Development Program of China (973
Program) (No. 2011CB504504) and the National
Science and Technology Pillar Program during the
Eleventh Five-year Plan Period (No. 2007BAI18B13).
References
1 Fransen E, Lemkens N, Van Laer L & Van Camp G
(2003) Age-related hearing impairment (ARHI): envi-
ronmental risk factors and genetic prospects. Exp
Gerontol 38, 353–359.
2 Bai U, Seidman MD, Hinojosa R & Quirk WS (1997)

Takeda S, Kaufman DG, Swenberg JA & Nakamura J
(2010) Accumulation of true single strand breaks and
AP sites in base excision repair deficient cells. Mutat
Res 694, 65–71.
11 Chen B, Zhong Y, Peng W, Sun Y, Hu YJ, Yang Y &
Kong WJ (2010) Increased mitochondrial DNA damage
and decreased base excision repair in the auditory cor-
tex of D-galactose-induced aging rats. Mol Biol Rep,
doi: 10.1007/s11033-010-0476-5.
Accumulation of mtDNA deletions in inner ear Y. Zhong et al.
2508 FEBS Journal 278 (2011) 2500–2510 ª 2011 The Authors Journal compilation ª 2011 FEBS
12 Kang D, Kim SH & Hamasaki N (2007) Mitochondrial
transcription factor A (TFAM): roles in maintenance
of mtDNA and cellular functions. Mitochondrion 7,
39–44.
13 Maniura-Weber K, Goffart S, Garstka HL, Montoya J
& Wiesner RJ (2004) Transient overexpression of mito-
chondrial transcription factor A (TFAM) is sufficient to
stimulate mitochondrial DNA transcription, but not
sufficient to increase mtDNA copy number in cultured
cells. Nucleic Acids Res 32, 6015–6027.
14 Ekstrand MI, Falkenberg M, Rantanen A, Park CB,
Gaspari M, Hultenby K, Rustin P, Gustafsson CM &
Larsson NG (2004) Mitochondrial transcription fac-
tor A regulates mtDNA copy number in mammals.
Hum Mol Genet 13, 935–944.
15 Canugovi C, Maynard S, Bayne AC, Sykora P, Tian J,
de Souza-Pinto NC, Croteau DL & Bohr VA (2010)
The mitochondrial transcription factor A functions in
mitochondrial base excision repair. DNA Repair (Amst)

24 Nicklas JA, Brooks EM, Hunter TC, Single R &
Branda RF (2004) Development of a quantitative
PCR (TaqMan) assay for relative mitochondrial
DNA copy number and the common mitochondrial
DNA deletion in the rat. Environ Mol Mutagen 44,
313–320.
25 Zhong Y, Hu YJ, Yang Y, Peng W, Sun Y, Chen B,
Huang X & Kong WJ (2011) Contribution of
common deletion to total deletion burden in mito-
chondrial DNA from inner ear of D-galactose-induced
aging rats. Mutat Res, doi: 10.1016/j.mrfmmm.
2011.03.013.
26 Markaryan A, Nelson EG & Hinojosa R (2009) Quanti-
fication of the mitochondrial DNA common deletion in
presbycusis. Laryngoscope 119, 1184–1189.
27 Trifunovic A, Wredenberg A, Falkenberg M, Spelbrink
JN, Rovio AT, Bruder CE, Bohlooly YM, Gidlof S,
Oldfors A, Wibom R et al. (2004) Premature ageing in
mice expressing defective mitochondrial DNA polymer-
ase. Nature 429, 417–423.
28 de Souza-Pinto NC, Eide L, Hogue BA, Thybo T, Stev-
nsner T, Seeberg E, Klungland A & Bohr VA (2001)
Repair of 8-oxodeoxyguanosine lesions in mitochondrial
dna depends on the oxoguanine dna glycosylase
(OGG1) gene and 8-oxoguanine accumulates in the
mitochondrial dna of OGG1-defective mice. Cancer Res
61, 5378–5381.
29 Evans MD, Dizdaroglu M & Cooke MS (2004) Oxida-
tive DNA damage and disease: induction, repair and
significance. Mutat Res 567, 1–61.

Genet 40, 275–279.
Y. Zhong et al. Accumulation of mtDNA deletions in inner ear
FEBS Journal 278 (2011) 2500–2510 ª 2011 The Authors Journal compilation ª 2011 FEBS 2509
37 Herbst A, Pak JW, McKenzie D, Bua E, Bassiouni M
& Aiken JM (2007) Accumulation of mitochondrial
DNA deletion mutations in aged muscle fibers: evidence
for a causal role in muscle fiber loss. J Gerontol A Biol
Sci Med Sci 62, 235–245.
38 Durham SE, Samuels DC & Chinnery PF (2006) Is
selection required for the accumulation of somatic mito-
chondrial DNA mutations in post-mitotic cells?
Neuromuscul Disord 16, 381–386.
39 Alam TI, Kanki T, Muta T, Ukaji K, Abe Y, Nakay-
ama H, Takio K, Hamasaki N & Kang D (2003)
Human mitochondrial DNA is packaged with TFAM.
Nucleic Acids Res 31, 1640–1645.
40 Hayashi Y, Yoshida M, Yamato M, Ide T, Wu Z,
Ochi-Shindou M, Kanki T, Kang D, Sunagawa K,
Tsutsui H et al. (2008) Reverse of age-dependent mem-
ory impairment and mitochondrial DNA damage in
microglia by an overexpression of human mitochondrial
transcription factor a in mice. J Neurosci 28,
8624–8634.
41 Ylikallio E, Tyynismaa H, Tsutsui H, Ide T & Suoma-
lainen A (2010) High mitochondrial DNA copy number
has detrimental effects in mice. Hum Mol Genet 19,
2695–2705.
42 Wanrooij S, Goffart S, Pohjoismaki JL, Yasukawa T &
Spelbrink JN (2007) Expression of catalytic mutants of
the mtDNA helicase Twinkle and polymerase POLG