ISSN 0006-2979, Biochemistry (Moscow), 2025, Vol. 90, No. 12, pp. 1849-1861 © Pleiades Publishing, Ltd., 2025.
1849
REVIEW
Turnover and Quality Control
of Mitochondrial DNA
Wolfram S. Kunz
Institute of Experimental Epileptology and Cognition Research, and Department of Epileptology,
University of Bonn, Venusberg-Campus 1, 53127 Bonn, Germany
e-mail: wolfram.kunz@ukbonn.de
Received August 7, 2025
Revised October 3, 2025
Accepted October 27, 2025
Abstract— The quantitative content of mitochondrial DNA (mtDNA) – a multicopy circular genome – is an
important parameter relevant for function of mitochondrial oxidative phosphorylation (OxPhos) in cells, since
mtDNA encodes 13 essential OxPhos proteins, 22 tRNAs, and 2 rRNAs. In contrast to the nuclear genome,
where almost all lesions have to be repaired, the multicopy nature of mtDNA allows the degradation of se-
verely damaged genomes. Therefore, cellular mtDNA maintenance and its copy number not only depend on
replication speed and repair reactions. The speed of intramitochondrial mtDNA degradation performed by
a POLGexo/MGME1/TWNK degradation complex and the breakdown rate of entire mitochondria (mitophagy)
are also relevant for maintaining the required steady state levels of mtDNA. The present review discusses
available information about the processes relevant for turnover of mitochondrial DNA, which dysbalance leads
to mtDNA maintenance disorders. This group of mitochondrial diseases is defined by pathological decrease of
cellular mtDNA copy number and can be separated in diseases related to decreased mtDNA synthesis rates
(due to direct replication defects or mitochondrial nucleotide pool dysbalance) or diseases related to increased
breakdown of entire mitochondria (due to elevated mitophagy rates).
DOI: 10.1134/S0006297925602485
Keywords: mtDNA maintenance, mtDNA replication, mtDNA degradation, determinants of cellular mtDNA con-
tent, mtDNA maintenance disorders
mtDNA REPLICATION MECHANISM
Human mitochondria contain a 16.5 kb circular
mitochondrial genome, which comprises the genetic
information for 13 proteins of the mitochondrial inner
membrane (subunits of OxPhos complexes), 22 tRNAs
and 2 rRNAs. In vitro experimental work showed that
the basic mitochondrial replisome required for repli-
cation of this genome consists of the mitochondrial
polymerase POLG, the mitochondrial single-stranded
DNA-binding protein (mtSSB), and the replicative he-
licase TWINKLE [1]. Although three different mod-
els of mitochondrial DNA (mtDNA) replication have
been proposed (for a comprehensive overview of all
three models cf. [1]), recent scientific literature fa-
vors the classical asynchronous strand-displacement
replication model of mtDNA [2], initially proposed by
the group of Vinograd [3] and later on verified by
experimental work of Clayton and coworkers [4, 5].
As shown in Scheme 1, according to this model rep-
lication is initiated at the OriH and replication of the
H-strand proceeds along 70% of the mtDNA until the
OriL is exposed and replication of the L-strand is
initiated and proceeds in the opposite direction [5].
Newer relevant data in support of this replication
model include (i) the conservation of OriL, (ii) the
occupancy of mitochondrial single-stranded DNA
binding protein, (iii) the mtDNA point mutation pro-
file, and (iv) the pattern of mtDNA ends detected by
ultra-deep long-read sequencing.
(i) CONSERVATION OF OriL
Using in vivo mutagenesis techniques Wanrooij
etal. [6] showed that OriL is indispensable for mtDNA
replication. When OriL is altered or deleted, mtDNA
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replication is severely impaired, underscoring its es-
sential function. The conservation and essentiality of
OriL strongly support the classical strand-displace-
ment model, as it shows that replication cannot pro-
ceed effectively without it.
(ii) In vivo OCCUPANCY OF SINGLE-STRANDED
DNABINDING PROTEIN
Fusté et al. [7] investigated how mtSSBs interact
with mtDNA during replication. mtSSBs selectively
bind to single-stranded regions of mtDNA in vivo,
particularly in the regions exposed during strand-dis-
placement replication. This binding stabilizes the
displaced single strand of DNA and protects it from
damage while replication proceeds on the opposite
strand. In the strand-displacement model, as the rep-
lication fork progresses, one strand of DNA becomes
temporarily single-stranded. The binding of mtSSBs
to these regions is a key feature, as it prevents deg-
radation or secondary structure formation in the dis-
placed strand. This study provides direct evidence for
mtSSBs’ role in maintaining strand stability in mito-
chondria, validating that strand-displacement occurs
and requires specific proteins to protect and to sta-
bilize the single-stranded regions.
(iii) mtDNA POINT MUTATION PROFILE
Recent research by Iliushchenko et al. [8] focused
on the mutation patterns within mtDNA across chor-
dates. The authors observed that some mutations are
linked to DNA damage, while others appear to arise
from replication-specific processes. For instance, regions
frequently exposed as single strands during strand-dis-
placement replication may accumulate mutations due
to increased vulnerability to damage or errors in rep-
lication. The presence of these replication-induced
mutation profiles aligns with the strand-displacement
model, as it implies that single-stranded regions are
exposed regularly enough to leave a mutational “sig-
nature.” This provides indirect support for the strand-
displacement mechanism by linking observed muta-
tion patterns to replication dynamics.
(iv) PATTERN OF mtDNA ENDS DETECTED
BYULTRA-DEEP LONG-READ SEQUENCING
Sensitive end detection by ultra-deep long-read
sequencing of native and nuclease S1-treated isolated
mtDNA confirmed in wild-type HEK293 cells the ab-
sence of Okazaki fragment related ends and provided
evidence for the presence of replication intermediates
having ends in the OriH, TAS, and OriL regions [9].
This result can be explained in the framework of the
classical replication model of mtDNA [3] only.
Together, these newer findings collectively af-
firm the asynchronous strand-displacement model
by showing how specific origins, stabilizing proteins,
replication-linked mutation patterns and detected
replication intermediates align with this particular
replication mechanism of mitochondrial DNA.
REPLICATION SPEED
AND TURNOVER MEASUREMENT OF mtDNA
Results from pulse-chase experiments indicate
that mtDNA in mouse L cells takes about 120 min to
Scheme 1. Strand-displacement model of mtDNA replication
(modified according to a scheme presented in [1]). 1) Repli-
cation of the nascent H-strand (dashed circle) is initiated at
OriH and proceeds unidirectionally by POLG/POLG2 (dark
blue) and unwinding by TWNK (light blue). In the process,
the parental H-strand is displaced and covered by mtSSB
(green). 2) When the replication machinery reaches OriL,
the H-strand of the origin folds into a stem-loop structure.
POLRMT (purple) initiates from the poly-dT stretch in the
loop region a short RNA primer (orange) that is used to ini-
tiate L-strand DNA synthesis. 3) The nascent L-strand is syn-
thesized continuously until full-circle and two new full-length
circular daughter molecules are formed. 4) Thesynthesis of
one daughter molecule containing the nascent H-strand is
initiated and terminated at OriH, whereas synthesis of the
other daughter molecule is initiated and terminated at OriL.
For removal of 5′ overhangs from the primers and formation
of ligatable ends MGME1 is required [89]. The end ligation
of the nascent strands is performed by LIG3 and at OriH
potentially also by TOP3A [9].
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Table 1. Half-life of mtDNA species in cell culture
Mitochondrial
constituent
Half-live Enzymes involved Methods References
mtDNA 7S DNA
7-9 h ? labelled nucleotides in mouse L-cells [12]
1 day MGME1 ddC-induced mtDNA depletion in fibroblasts [49]
4 days ?
ddC-induced mtDNA depletion
in MGME
–/–
fibroblasts
[49]
Linear mtDNA
fragments
2-4 h MGME1/POLGexo fragment analysis in HEK cells [37]
Total mtDNA 8-12 days* ? labelled nucleotides in epithelial cells [13]
Note. * Can be influenced by re-use of nucleotides.
complete one replication cycle [10]. This is relatively
fast, representing only about 5% of the entire cell cy-
cle. Unlike nuclear DNA that replicates synchronous
with the cell cycle, mtDNA replication is not tightly
coupled with nuclear DNA replication. This means
mtDNA can replicate at different times, independent
of the cell’s nuclear DNA replication schedule, al-
though imaging studies of synchronized HeLa cells
detected that initiation of replication is coordinated
with the cell cycle, preceding nuclear DNA synthe-
sis [11]. The asynchronous replication allows mito-
chondria to produce more mtDNA whenever needed,
which might be crucial for energy-demanding condi-
tions in the cell. Interestingly, the 7S heavy-strand of
mtDNA initiates more frequently, as indicated by a
short half-life of 7-9 h [12]. It is part of a unique re-
gion in mtDNA called the D-loop, which is critical for
starting mtDNA replication.
Labelling studies with
3
H-labelled nucleosides
show that the half-life of mtDNA in relatively short-
lived cells, such as epithelial cells or hepatocytes, is
8-12 days [13], whereas, in certain post-mitotic cells,
such as neurons, the half-life of the mtDNA is 20-30
days. Postmitotic heart cells are an exception with a
mtDNA half-live of 6-7 days [14]. Interestingly, under
the investigated labeling conditions of heart cells no
measurable turnover of nuclear DNA was noticed.
In the classical literature it has been reported
that entire mitochondria show tissue-specific rates
of turnover, if assessed by turnover of mitochondrial
proteins, lipids or mtDNA. For example, in the mouse
heart, mitochondria turn over with a half-life of 14
days [15], but in the liver, the half-life is 2-4 days [15,
16] and in brain 26 days [15]. This can be explained
by selective mitochondrial autophagy, or mitophagy,
which eliminates damaged and dysfunctional mito-
chondria [16-18] and is closely linked to mitochondrial
biogenesis, which permits replacement of mitochon-
dria (or synthesis of components and their insertion
into the remaining functional mitochondria). Longer
half-life of mtDNA in comparison to proteins might
result from a re-use of the labelled nucleotides (cf.
Table 1). The older literature data (cf. [15]) are in gen-
eral agreement with the original hypothesis of Fletcher
and Sanadi, who initially proposed mitophagy as main
contributor to turnover of mitochondria. However,
mitophagy of entire mitochondria obviously cannot
explain the relatively large (approximately 10-fold)
differences of turnover rates of individual proteins of
the mouse proteome as determined by more advanced
MS techniques using metabolic heavy water (
2
H
2
O) la-
beling [19]. Asexamples in mouse heart ATP5i and CS
had a half-life of 54.1 days and 41.3 days, respective-
ly, while OAT and DNAJC30 of 3.3 days and 4.4 days,
respectively. In mouse liver the half-life of ATP5i and
CS was 8.5 days and 7.5 days, respectively and of OAT
1.9 days [19]. With MS techniques remarkable differ-
ences in turnover were even detected for individual
subunits of respiratory chain complex I: proteins of
the N-module showed elevated turnover in compari-
son to the Q-module or the mitochondrially encoded
subunits, which was strongly affected by mutations
in the chaperone protein DNAJC30 [20].
DETERMINANTS OF mtDNA CONTENT IN CELLS
The mtDNA content in yeast cells as determined
by a study of Seel et al. [21] depends on cell size,
which directly influences the mtDNA amount which
can change from 20 to 160 copies per cell. As cells
grow, mitochondrial biogenesis is regulated to en-
sure mtDNA homeostasis, maintaining a consistent
ratio relative to cell volume. This regulation ensures
that energy production capacity meets cellular de-
mands during growth. With cell size correlates the
expression of the mitochondrial DNA polymerase
Mip1 (the yeast POLG paralogue) and the yeast TFAM
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paralogue Abf2 [21]. In yeast cells, other factors in-
volved in mtDNA replication, like the helicase or the
ssDNA-binding protein Rim1 had smaller effects.
In mammals, which contain a much more com-
pact mitochondrial genome, the copy number of
mtDNA (the mtDNA/nDNA ratio) is highly variable
and is strongly cell type dependent: human sperm
cells contain as little as 5-10 copies of mtDNA per nu-
clear genome [22] and in a recent study using digital
droplet PCR even only 0.58 copies per nucleus were
detected [23], which was attributed to a lack of func-
tional TFAM. On the other hand, human oocytes may
contain as many as 500,000 copies of mtDNA [24].
Overexpression of either Twinkle or TFAM in
mice leads in mammals to a higher mtDNA amount
and enhances recovery from ischemic heart damage
[25-28] or delays of male infertility [29]. On the oth-
er hand, overexpression of DNA polymerase gamma
(POLG), the mtDNA replicase, in contrast to yeasts
does not affect mtDNA copy number in mammals,
emphasizing the controlling role of the helicase
Twinkle and TFAM for mammalian mtDNA replica-
tion and thus the cellular mtDNA content.
In addition to the replication process the copy
number of mtDNA (the mtDNA/nDNA ratio) is obvi-
ously directly affected by biosynthesis of mitochon-
dria. Here, nuclear transcription factors and coactiva-
tors play a critical role in mitochondrial biogenesis,
particularly those in the PGC-1 (Peroxisome Prolifer-
ator-Activated Receptor Gamma Coactivator 1) fam-
ily, as highlighted by Scarpulla [30, 31] and others.
These nuclear factors integrate signals that promote
mtDNA replication and transcription, adapting mi-
tochondrial function to meet energy requirements.
PGC-1α integrates the activity of many transcription
factors, including NRF1, NRF2, and ERRα [30-32]. The
expression of PGC-1α is significantly induced upon
oxidative stress, in turn enhancing the expression
of some antioxidant proteins, which can prevent ex-
cessive mitochondrial ROS production following mi-
tobiogenesis [33]. Environmental stimuli, like cold
exposure or training conditions can drive mitochon-
drial biogenesis. Wu et al. [34] describe how PGC-1
coactivators trigger thermogenic responses in brown
adipose tissue, increasing mtDNA content and overall
mitochondrial capacity as an adaptation to increased
energy needs. Here, PGC-1α increases the transcrip-
tional activity of NRF1, stimulates the production
of NRF1 and NRF2, and increases the expression of
TFAM and numerous mitochondrial respiratory chain
genes, leading to biogenesis of mitochondria [34].
Thus, the cellular mtDNA content is tightly con-
trolled by both intrinsic factors, like cell size, mtDNA
replication speed and nuclear regulatory proteins,
and extrinsic factors, such as environmental and met-
abolic stresses.
INTRAMITOCHONDRIAL
mtDNA DEGRADATION
Due to the redundant nature and multiple copies
of mtDNA, it is likely that intramitochondrial degra-
dation of damaged mtDNA is the most efficient path-
way to cope with double strand breaks (DSBs) or an
overwhelming number of mutagenic lesions [35]. This
is most probably the reason that mtDNA is rapidly
degraded after extensive oxidative stress [36]. Inter-
estingly, mtDNA replication proteins were found to
be not only required for synthesis but also involved
in the degradation of linear mtDNA fragments, spe-
cifically the 5′-3′ exonuclease MGME1 and the 3′-5′
exonuclease of POLG [37]. Since only blunt end dou-
ble-stranded degradation intermediates have been
identified after induction of mitochondrially targeted
restriction enzymes [37] leading to defined DSBs, it
has been proposed that both exonucleases – preferen-
tially digesting single-stranded DNA molecules in sep-
arate directions – collectively work together on both
strands. Interestingly, in all experiments which lead
to the induction of DSBs in mtDNA blunt-end double
stranded degradation intermediates with ends proxi-
mal to GC-rich regions have been identified [37, 38].
Degradation of mtDNA after DSBs, as opposed
to end joining repair pathways present in the nucle-
us, avoids the formation of deletions [37, 39]. Intact
mtDNA copies are then replicated to repopulate wild-
type mtDNA species [40]. This has led to the idea of
mtDNA being a “disposable” genome due to the multi-
ple copies and the ability to repopulate independent-
ly of cell cycle [41], because degradation combined
with the replication of intact copies is more efficient
to maintain intact mtDNA rather than to invest in an
error-prone repair [42]. Accordingly, all of the con-
stituents of base-excision repair pathway have been
identified in mitochondria [40], while double-strand
break repair activities are apparently restricted to
microhomology-mediated end joining only [43], which
are implicated in the formation of pathogenic mtDNA
deletions.
As stated above, the intramitochondrial degrada-
tion of linear dsDNA has been shown to depend on
the action of two ssDNA specific exonucleases – the
3′-5′ exonuclease of POLG [37, 39] and MGME1 [37] –
an exonuclease showing apparently a bidirectional
ssDNA splitting behavior [44, 45]. Residual dsDNA
splitting activity of MGME1 can be ruled on the basis
of nuclease assays [44, 45] and its molecular mech-
anism – a ring-shaped architecture with a selective
hole that allows only ssDNA to access the catalytic
center [46]. For MGME1 a higher 5′-3′ activity with
ssDNA substrates has been initially observed by clas-
sical nuclease assays [44, 47] and an approximately
3-fold 5′-3′ over 3′-5′ activity has been convincingly
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confirmed by a fluorescence-based single molecule
method [48]. To explain efficient degradation of long
linear dsmtDNA fragments leading to the transient
generation of shorter linear blunt-end dsDNA inter-
mediates the action of two ssDNA specific enzymes
with distinct direction preferences would be required.
The concerted action of both exonucleases – MGME1
and POLGexo – as proposed in [37], fulfills these re-
quirements. That also explains experimental data for
additional requirement of the mitochondrial helicase
TWINKLE [37], needed for unwinding of long linear
dsDNA fragments.
Notably, a physical protein–protein interaction
between a small fraction of POLG and MGME1 has
been reported by high-resolution label-free mass
spectroscopic and pull-down approaches in MGME1
overexpressing HEK293T cells [49]. These results [49]
and the surprising observation that the lentiviral res-
cue of MGME1 in MGME1-deficient fibroblasts (which
leads to a severe MGME1 overexpression) does not
normalize mtDNA copy numbers, but causes severe
mtDNA depletion [44] are compatible with the spec-
ulation that particularly the recruitment of MGME1
to POLG forms a degradation complex, suitable for
efficient degradation of double-stranded mtDNA. The
hypothetical detail of molecular events describing the
degradation of EagI- or PstI-linearized mtDNA (as de-
scribed in detail in [37]) or linear mtDNA fragments
occurring after oxidative stress [38] can be outlined
as follows:
1. DSB-lesion detection by POLG
2. Filling-in of short 5′ overhangs (of note: these
overhangs are not degraded by MGME1!) or deg-
radation of short 3′ overhangs by POLG to gen-
erate blunt ends
3. Recruitment of MGME1 and TWNK to the blunt
ends, respectively
4. Formation of a degradation complex between the
three proteins
5. Degradation of linear mtDNA with transient gen-
eration of blunt-end dsDNA intermediates due to
the slowing down of degradation activity at GC-
rich stretches
This hypothetical mechanism of degradation of
linear mtDNA is presented in Scheme 2. The pres-
ence of degradation stop-sites proximal to GC-rich
regions of mtDNA [37, 38] can be explained by the
elevated double-strand melting temperature at these
positions (GC pairing involves 3 hydrogen bonds ver-
sus 2 hydrogen bonds by AT pairing), which should
slow down the helicase activity of TWNK, being also
rate-limiting for replication [50]. Additionally, also
the degradation speed of MGME1 is influenced by
the nucleotide composition of the ssDNA fragments:
GC-rich fragments show the lowest degradation speed
[47]. Quite recently, direct evidence has been provid-
ed that even intrinsic oxidative stress is relevant for
mtDNA turnover in HEK293 cells [8], since knock-out
of LIG3 leads to accelerated mtDNA degradation due
to inability of repair of intrinsic oxidative lesions.
MITOPHAGY
As mentioned above in “Replication speed and
turnover measurement of mtDNA” section, mtDNA
can be also removed from cells by degradation of
mitochondria as entire organelle. This process named
mitophagy has been described in the literature as
specific autophagic elimination of entire mitochon-
dria. The concept of mitophagy has been originally
proposed by Fletcher & Sanadi [15] as main principle
to explain the concerted turnover of mitochondrial
proteins and phospholipids observed in hepatocytes.
In mammals, mitophagy is generally divided into
PINK1/Parkin-dependent or canonical mitophagy, and
PINK1/Parkin-independent mitophagy. The PINK1/Par-
kin-dependent pathway can be initiated by a loss of
mitochondrial membrane potential [51], while PINK1/
Parkin-independent mitophagy does not require
loss of the mitochondrial membrane potential [52].
Scheme 2. Hypothetical mechanism of mtDNA degradation
after double strand breaks involving a degradation com-
plex consisting of MGME1, POLGexo, and TWNK (modified
according to the scheme presented in [37]). Importantly,
binding of POLG2 to POLG is not required for the formation
of the degradation complex and a contribution of other mi-
tochondrial nucleases (EXOG, APEX2, ENDOG, FEN1, DNA2,
MRE11, or RBBP8) was excluded by knock-out or knock-
down experiments [37].
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Table 2. Processes to be considered relevant for defining the mtDNA copy number in cells
Speed Enzymes/proteins involved References
Intramitochondrial process
Replication 2 h POLG, POLG2, TWNK, mtSSB [11]
Degradation of linear
mtDNA
~8 h* POLG, MGME1, TWNK [37]
Extramitochondrial process involving singular organelles or MDVs
Biogenesis
Synchronized
with cell cycle
PGC1-α, Nrf1, Nrf2, Err-α, TFAM [31, 32]
Mitophagy (PINK1/Parkin
dependent)
4-24 h PINK1/PARKIN [51, 67, 68]
Mitophagy (PINK1/Parkin
independent)
? BNIP3/BNIP3L or FUNDC1 or FKBP8 or BLC2L13 [52]
Autophagy of MDVs ? FUNDC1 [59]
Note. MDV – mitochondria-derived vesicles. * Time required for complete mtDNA degradation after induction of a double-
strand break by mtEagI.
PINK1/Parkin-dependent and -independent pathways
utilize different sets of autophagy receptors [53]. Itis
assumed that the autophagy receptors involved in
PINK1/Parkin-dependent mitophagy are cytosolic pro-
teins that interact with ubiquitinated proteins of the
outer mitochondrial membrane via ubiquitin-binding
domains. Receptors involved in PINK1/Parkin-inde-
pendent mitophagy are integral mitochondrial pro-
teins – like BNIP3 and BNIP3L (NIX) – that interact
directly with ATG8 family proteins using the general
macroautophagy/autophagy machinery [53]. The lat-
ter form of mitophagy has been reported to be im-
portant for mtDNA quality control in the germline of
Drosophila [54].
From a mechanistic perspective a prerequisite
for mitophagy are singular organelles, thus mito-
chondrial fission obviously should precede degrada-
tion. Indeed, early evidence points toward mitochon-
drial fission being important for mitophagy [55-57],
thus, supporting a direct link between mitochondrial
fragmentation and mitophagy. Similarly, the axonal
transport machinery for mitochondria in neurons es-
sentially requires fragmented mitochondria as cargo
[58]. However, the LIR-containing cytosolic portion of
FUNDC1 is sufficient to induce mitophagy even in the
absence of mitochondrial fragmentation [59], indicat-
ing that PINK1/Parkin-independent mitophagy can be
uncoupled from mitochondrial morphology. Indeed,
mitophagy still occurs in the absence of DRP1 in the
presence of other mitophagy inducers like hypoxia,
iron chelation, or proteotoxic stress [60, 61]. In such
cases, a vesicle-like budding of the mitochondria has
been proposed to contribute to the generation of
small mitochondrial fragments that can be engulfed
for the mitophagic process [62]. These mitochondri-
al fragments are very likely identical to the so-called
MDVs described to be relevant for quality control of
the cardiac system [63]. These vesicles can contain
mtDNA and therefore also contribute to mtDNA degra-
dation by the autophagic removal of leaked damaged
mtDNA [64] or even entire nucleoids [65]. Therefore,
the before mentioned quite large differences in the
turnover times of individual mitochondrial proteins
[19] and also of mtDNA (cf. “Replication speed and
turnover measurement of mtDNA” section) do not ex-
clude a substantial contribution of autophagy in turn-
over, since the vesicles could contain different protein
and mtDNA fractions.
Studies indicate that in neurons, the sequestra-
tion of damaged mitochondria into autophagosomes
can occur within approximately 1-2 h. However, the
subsequent acidification and degradation within ly-
sosomes is significantly delayed, often exceeding 6 h,
and in some cases, extending beyond 24 h [66]. This
suggests that the degradation phase is a rate-limiting
step in neuronal mitophagy. In contrast, non-neuro-
nal cells like HeLa cells exhibit a more rapid progres-
sion. The entire mitophagy process, from initiation to
degradation, can be completed within 4-24 h, with ly-
sosomal degradation occurring more swiftly than in
neuronal cells [67, 68] (cf. Table 2). However, in this
time scale mitochondrial density markers (like TRME
or mitotracker fluorescence) did not change signifi-
cantly [67], indicating that in these studies reporting
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a fast autophagic process only very limited amounts
of mitochondria were involved.
mtDNA COPY NUMBER AND DISEASE
Decreased copy numbers of mitochondrial DNA
in human tissue samples are the diagnostic hallmark
of so-called mitochondrial DNA maintenance disor-
ders – a heterogeneous subgroup of mitochondrial
diseases (Table 3). Here, the mtDNA copy number is
defined as ratio of mtDNA copies per nucleus in the
sample. Obviously, it depends on two factors: (i) the
mitochondrial content of tissue and (ii) the mtDNA
content of the mitochondrial network. As explained
above (cf. “Determinants of mtDNA content in cells”
section) the mitochondrial content of the tissue is
dependent on metabolic needs, like energy require-
ments which affect expression of PGC-1α facilitat-
ing biosynthesis of mitochondria and on the break-
down rate of damaged mitochondria by mitophagy
(cf. “Mitophagy” section). This value is obviously
strongly tissue dependent with the highest values in
cardiac and skeletal muscle and low values in sperm
cells (cf. “Determinants of mtDNA content in cells”
section). On the other hand, mtDNA is packed within
nucleoids and the nucleoid content of the mitochon-
drial network is in the range of 300-800 nucleoids per
cell [69] and each nucleoid contains about 1 copy of
mtDNA [70].
Pathological deviations of cellular mtDNA con-
tent are described in following conditions:
1. Decreased mtDNA copy number due to decreased
rate of replication (mutations in POLG, POLG2,
TWNK, MGME1, mtSSB). This ultimately leads to
low levels of mtDNA causing reduced synthesis
of mtDNA-encoded proteins and strong impair-
ment of OxPhos.
2. Decreased mtDNA copy number due to decreased
rate of mtDNA replication related to nucleotide
dysbalance (mutations in TK2, DGUOK, ANT1,
TYMP, SUCLA2, SUCLG1, MPV17). Since dysbal-
ance of mitochondrial desoxyribonucleotide tri-
phosphates directly affects replication speed of
POLG this causes similar to (1)lowered synthesis
of mtDNA-encoded proteins and strong impair-
ment of OxPhos. While most of the mentioned
genes are directly related to the mitochondrial
synthesis of phosphorylated desoxyribonucleo-
tides, MPV17 forms an ion channel which trans-
locates uridine but not orotate across the mito-
chondrial inner membrane [71].
3. Decreased mtDNA copy number due to elevat-
ed degradation of mitochondria by mitophagy
(mutations in FBXL4, MFN2, OPA1). This leads
to a lowered content of mitochondria and
Table 3. Mitochondrial diseases caused by alterations
of the mtDNA copy number (mtDNA maintenance dis-
orders)
Affected
gene
Clinical phenotype References
Diseases due to mutated proteins involved
in replication
POLG muscular or brain/liver
phenotype, recessive
and dominant
[72, 73]
POLG2 recessive and dominant [74]
TWNK muscular (CPEO) or brain
(ataxia) phenotype, dominant
and recessive
[75]
MGME1 multisystemic phenotype,
recessive
[44]
mtSSB optic atrophy, dominant [76]
Diseases due to mutated proteins involved
in maintenance of the nucleotide pool
TK2 muscular phenotype,
recessive
[77]
DGUOK hepatocerebral and muscular
phenotype, recessive
[78]
TYMP neurogastrointestinal
encephalopathy, recessive
[79]
ANT1 cardiomyopathic phenotype,
recessive and dominant
[80]
SUCLA2 encephalomyopathic
phenotype, recessive
[81]
SUCLG1 encephalomyopathic
phenotype, recessive
[82]
MPV17 hepatocerebral phenotype,
recessive
[83]
Diseases due to mutated proteins involved
in mitophagy or mitochondrial dynamics
FBXL4 encephalomyopatic
phenotype, recessive
[84]
MFN2 neuropathy of peripheral
nerves, dominant
and recessive
[85, 86]
OPA1 optic atrophy, dominant
and recessive
[87]
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consequently also of mtDNA in single cells and
the tissue, however no specific OxPhos impair-
ment can be observed. That condition affects cel-
lular metabolism particularly in cells relying on
OxPhos for energy production or the supply of
precursor metabolites.
Under the conditions #1 and #2 obviously the
synthesis rate of mtDNA is decreased. That leads at
a constant breakdown rate of mtDNA to decreased
steady state levels of mtDNA at an almost unchanged
cellular content of mitochondria. As consequence a
reduced transcription and translation of mtDNA-
encoded proteins is observed. In contrast, condition
#3 decreases the total cellular content of mitochondria
due to an increase of speed of mitophagy as in case
of FBXL4 mutations, which disrupt the post-transla-
tional regulation of BNIP3L and BNIP3 [88]. Similarly,
mutations in proteins involved in the fusion process
of mitochondrial membranes like MFN2 (outer mem-
brane fusion) or OPA1 (inner membrane fusion) lead
to a higher fragmentation of mitochondria, which also
facilitates mitophagy, thus explaining reduced mtDNA
copy numbers, reported in [86] and [87]. Since the
reduction of mtDNA is associated under these con-
ditions with a reduced cellular content of mitochon-
dria, the effects on transcription and translation of
mtDNA-encoded proteins are only very modest.
As can be seen in Table3, the phenotypes detect-
ed in mtDNA maintenance disorders are as variable
as reported for other mitochondrial disorders, rang-
ing from severe encephalomyopatic to milder neu-
ropathic or myopathic phenotypes. Genotype-pheno-
type correlations can be only very rarely made, since
similar mutations can result, like reported for POLG
mutations [72, 73], in distinct phenotypes, which is
potentially related to different genetic backgrounds of
the affected patients.
CONCLUSIVE REMARKS
In contrast to the nuclear genome, where almost
all lesions – including DSBs – have to be repaired, the
multicopy nature of mtDNA allows the degradation of
severely damaged genomes, containing DSBs, to avoid
deletion formation. This feature is responsible for a
steady turnover of mitochondrial DNA, which is very
relevant for mtDNA quality control in postmitotic
cells. Therefore, cellular mtDNA maintenance and its
copy number not only depend on replication speed
and repair reactions. The speed of intramitochon-
drial mtDNA degradation performed by a POLGexo/
MGME1/TWNK degradation complex and the break-
down rate of entire mitochondria (mitophagy) are
also highly relevant for maintaining required steady
state levels of intact mtDNA.
Abbreviations
DSB double strand break
mtDNA mitochondrial DNA
mtSSB mitochondrial single-stranded
DNA-binding protein
OxPhos oxidative phosphorylation
OriH heavy strand replication origin
OriL light strand replication origin
SSB single strand break
Acknowledgments
This work is dedicated to the 90th birthday of Prof.
Vladimir Petrovitch Skulachev, who was the PhD the-
sis supervisor of the author from 1980-1983.
Funding
This work was financially supported by a grant from
the DFG – Deutsche Forschungsgemeinschaft (grant
KU911/22-1).
Ethics approval and consent to participate
This work does not contain any studies involving hu-
man and animal subjects.
Conflict of interest
The author of this work declares that he has no con-
flicts of interest.
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