Download Clinical and Molecular Aspects of Diseases of Mitochondrial DNA

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354
Clinical and Molecular Aspects of Diseases of Mitochondrial
DNA Instability
Chih-Chieh Mao, MD, PhD; Ian J. Holt1, PhD
Mitochondria within human cells contain vast numbers of mitochondrial DNA
(mtDNA), which are small, circular, and double-stranded. The proper functions of mtDNA
depend totally on specific proteins that are encoded by the nucleus and then imported into
mitochondria. Thus instability of mtDNA can stem from the mtDNA itself, or secondarily
from abnormalities in nuclear DNA. In this review, we will first introduce mtDNA, including its characteristics, replication, transcription, translation, and the proteins involved in its
metabolism, in particular DNA polymerase γ (POLG), DNA helicase Twinkle (Twinkle),
and mitochondrial transcription factor A (TFAM). Secondly, we will stress the importance of
mitochondrial nucleoid structures in the protection and facilitation of mtDNA metabolism,
and report on the few known protein components of nucleoid, especially Twinkle, TFAM,
and the recently discovered ATAD3. Based on this information, mtDNA instabilities will be
categorized in accordance with their molecular etiologies, those that are caused by primary
defects of mtDNA, and those by secondary effects from abnormalities in nuclear DNA. The
former includes large defects or point mutations of mtDNA. The latter involves the nuclear
genes of POLG1, Twinkle, ANT1, TK2, dGK, and TP. With the comprehensive categorization in this review, links are provided between the molecular and clinical aspects of mitochondrial DNA diseases. This report should help medical staff understand the complexity of
these diseases and encourage them in further investigations. (Chang Gung Med J
2009;32:354-69)
Key words: mitochondrial DNA, mitochondrial nucleoid, mitochondrial diseases
1. General features of mitochondria
M
itochondria are subcellular organelles within
the cytoplasm of nearly all eukaryotic cells (a
rare exception is the erythrocyte), which usually constitute ~25% of the volume of cytoplasm. They are
not static entities. Instead they move along the
cytoskeletal network,(1,2) undergoing frequent fusion
and fission.(3-5) When cells divide, mitochondria are
shared almost equally among the daughter cells.
Mitochondria have an outer membrane and an inner
membrane separated by a cavity, the intermembrane
space.(6) The outer membrane is highly permeable,
containing many pores for small molecules to pass
through. In contrast, the inner mitochondrial membrane (IMM) is impermeable to most small molecules and ions, a property required for maintenance
of an electrochemical gradient.(7) The IMM folds into
lamellar structures called cristae,(6) which increase its
surface area and mass. It is estimated that three quarters of the mass of the IMM is protein, and many of
these proteins are components of the oxidative phosphorylation system (OXPHOS). The interior com-
From the Department of Anesthesiology, Chang Gung Memorial Hospital, Taipei, Chang Gung University College of Medicine,
Taoyuan, Taiwan; 1Medical Research Council Dunn Human Nutrition Unit, Cambridge, U.K.
Received: Jun. 17, 2008; Accepted: Dec. 26, 2008
Correspondence to: Dr. Chih-Chieh Mao, Department of Anesthesiology, Chang Gung Memorial Hospital. 5, Fusing St., Gueishan
Township, Taoyuan County 333, Taiwan (R.O.C.) Tel.: 886-3-3281200 ext. 2324; Fax: 886-3-3281200 ext. 2787;
E-mail: [email protected]
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partment of mitochondria is composed of a gel-like
substance, the matrix, which contains enzymes of the
tricarboxylic acid (TCA) cycle, and β-oxidation.
These oxidation-reduction reactions and energy
transfer processes make mitochondria the powerhouses of eukaryotic cells.
The above chemical reactions (TCA cycle, βoxidation, and OXPHOS) involve a large number of
proteins in the mitochondria. It is estimated that the
mitochondrion contains approximately 1000 proteins, of which only 3-32, depending on the organism, are encoded by mtDNA.(8) The remainder are
encoded by nuclear DNA, translated in the cytosol
and imported into mitochondria via translocases of
the outer membrane (TOM complex) and inner membrane (TIM22, TIM23 complexes).
2. Mitochondrial DNA
Mitochondria, like chloroplasts, have their own
DNA, mitochondrial DNA (mtDNA). However,
mtDNA is itself dependent on nuclear genes for its
replication, expression, and maintenance (see sections 3, 4 & 5 of this report). In mammals, mtDNA
are covalently closed circular molecules of doublestranded DNA interspersed with a handful of ribonucleotides on each strand.(9) In a single cell the copy
number of mtDNA is typically over 1,000. This huge
number of mtDNA molecules is organized into discrete foci called mitochondrial nucleoids (mtnucleoids; see sections 5 and 6), which are believed
to be tethered to the IMM. Mt-nucleoids comprise
multiple copies of mtDNA and an unknown number
of proteins. Nuclear DNA encodes all the proteins
involved in mt-nucleoid metabolism. Most of the little that is known about mammalian mt-nucleoids has
come from studies of human diseases caused by
mtDNA perturbation (section 10).
There is little variation in the size of mtDNA in
animals; the range is approximately 16-19 kb. The
genes are tightly packed; there are no introns, many
genes are contiguous, and some even overlap.
Mammalian mtDNA differs from nuclear DNA
in several ways. It is small, circular, has a high copy
number per cell, and is inherited through the maternal line. The two strands of mtDNA are separable on
alkaline caesium chloride gradients, owing to a bias
in the nucleotide composition. The so-called heavy
strand (H-strand) is rich in guanines; while the light
strand (L-strand) is rich in cytosines. The human
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mitochondrial genome is 16,569 base pairs (Fig. 1).
Despite its small size, mtDNA encodes 22 transfer
RNAs, 2 ribosomal RNAs and 13 polypeptides
which are required for OXPHOS. Although the number 1000 is widely quoted as the mtDNA complement of a typical cell, there is in fact considerable
variation in the mtDNA copy number among different cell types and tissues. The key determinant of the
copy number is energy demand; thus, cardiac muscle
has a considerably higher mtDNA copy number than
skin cells. A high mtDNA copy number appears to
protect against defects in mtDNA because OXPHOS
can still be maintained without a significant cellular
phenotype, until or unless a large proportion of
mtDNA molecules is defective.
Animal oocytes contain an extraordinary number of mtDNA molecules, about 100,000 in frogs and
humans. In contrast to oocytes, spermatozoa contain
few mitochondria, even less than a typical epithelial
cell, although there is no doubt that their function is
required for sperm motility. Mitochondria of sperm
are located in the mid-piece, which is actively
degraded upon entry into the egg.(10) Immediately following fertilization, mtDNA replication is quiescent,
and is only activated at the 2-cell stage in mouse
embryos.(11) The degradation of sperm mitochondria
and the preponderance of oocyte mtDNAs ensure
strict maternal inheritance of mtDNA.
Human mtDNA is compact and economical,
managing to compress 37 genes into fewer than 20
kilobases of DNA. There is, however, one substantial
non-coding region (NCR), of ~900 bp in mammals
(Fig. 1). Within the NCR, many molecules of
mtDNA contain a triple-stranded region, or displacement loop (D-loop). (12-14) The third strand, or 7S
DNA, is synthesised via a transcription event initiating at the light strand promoter (LSP), and then a
transition to DNA synthesis at the origin of heavy
strand replication (OH). In humans, the size of 7S
DNA is about 650 bp, spanning nucleotide numbers
16,111 to nucleotide 191. The purpose of the D-loop
is a matter of conjecture.
3. Mitochondrial DNA replication
The mitochondrial DNA copy number is related
to energy demand: tissues with the highest respiratory demand, such as muscle, liver, brain, and pancreatic islets, have the greatest number of copies of
mtDNA. Therefore replication of mtDNA not only is
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HSP2
OH
HSP
F
12S rRNA
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T
NCR
V
Cyt b
P
LSP
16S rRNA
E
ND6
ND5
MELAS 3243G
LUUR
LHON 14484C
LCUN
ND1
LHON 3460A
SAGY
H
16,569 bp
I
LHON 11778A
Q
ND4
NARP T9176G/C
M
ND4L
NARP 8993G/C
QL
ND2
N
ND3
G
COX3
MERFF 8344G
A
W
R
C
Y
ATPase 6
SUCN
COX1
K ATPase 8
D
COX2
5 kb
deletion
Complex I genes
Complex III genes
Transfer RNA genes
Complex IV genes
ATPase genes
Ribosomal RNA genes
Fig. 1 Schematic representation of 16,569 base-pair circular human mitochondrial DNA (mtDNA) showing genes encoding subunits of complex I, III, IV, and ATPase; ribosomal RNAs, and transfer RNAs according to the single letter code of the amino acids.
The heavy strand promoter (HSP), light strand promoter (LSP), origin of heavy strand replication (OH), and a second heavy strand
promoter (HSP2) are marked by bent arrows. The origin of lagging strand synthesis (OL) in a short spacing region is also marked by
a bent arrow. Common pathogenic mutations (NARP, LHON, MERFF, MELAS) associated with mitochondrial disease are indicated by arrows. A large scale partial deletion (nucleotide position 8469 to 13447), seen in one third of patients with PEO/KSS/PS, is
marked as a 5 kb deletion. Abbreviations used: NARP, neurogenic muscle weakness, ataxia, and retinitis pigmentosa syndrome;
LHON, Leber’s hereditary optic neuropathy syndrome; MERFF, myoclonus epilepsy with ragged-red fibres syndrome; MELAS,
mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes syndrome; PEO/KSS/PS, progressive external ophthalmoplegia/Kearns-Sayre syndrome/Pearson’s syndrome.
fundamental to cell biogenesis, but should also be
regulated by the metabolic status of a given tissue at
different stages of development or diseases.
3.1. Mechanisms of mtDNA replication
There are two competing models of mtDNA
replication in mammals, strand-asynchronous repli-
cation, and coupled leading and lagging strand replication. In the strand-asynchronous model, the 2
strands of mtDNA initiate DNA synthesis in a temporally asynchronous way from 2 spatially separate
points on the mtDNA, termed heavy (OH) and light
(OL) strand origins. H strand replication is primed
from a processed transcript initiated at the LSP with-
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in the NCR. The transcript is terminated at another
region within the NCR known as the conserved
sequences blocks (CSBs) and then processed, thereby creating a primer for DNA synthesis; the RNADNA transition point defines the origin of heavy
strand replication (OH, typically nucleotide 191 of
human mtDNA). H-strand DNA synthesis proceeds
until it has traversed two-thirds of the genome,
whereupon it exposes a short spacer region which is
theoretically capable of forming a stem loop structure; separation of the two strands allows initiation of
DNA synthesis of the second strand to commence.(15)
This mechanism predicts extensive single-stranded
regions in replicating molecules. Recently the model
has been revised and alternative light strand ‘origins’
have been proposed.(16)
Over the past few years an alternative view of
mtDNA replication has been elaborated.
Mitochondrial DNA molecules separated by twodimensional agarose gel electrophoresis have the
appearance and properties of duplex replication
intermediates. A fraction of these have all the hallmarks of products of conventional coupled leading
and lagging strand synthesis.(17) However the majority are sensitive to RNase H(18) and this has led recently to the proposal that RNA is incorporated throughout the lagging strand at high frequency during
mtDNA replication, the RITOLS mechanism.(19)
ty, and initiation and elongation of DNA replication.(25)
Mitochondria also contain a DNA helicase
called Twinkle, which is related to the primase-helicase of T7 phage. (26) The helicase domain is conserved; however, the primase domain of T7 primasehelicase has no obvious similarity to Twinkle.
Recombinant Twinkle has a DNA helicase activity
with 5’ to 3’ directionality in vitro, and DNA
unwinding by Twinkle is specifically stimulated by
mtSSB.(27) In vitro, Twinkle, POLG, and mtSSB form
processive replication machinery, which can use double-stranded DNA as a template to synthesize singlestranded DNA molecules of about 16 kb.(28)
Mitochondrial DNA is supercoiled in vivo and
its replication, as in other DNA systems, requires
topoisomerases to relax and wind up DNA molecules. Mitochondrial topoisomerase 1 (TOP1MT) is
homologous to nuclear topoisomerase 1, except that
it has an N-terminal mitochondrial targeting signal in
place of a nuclear localisation sequence.(29) TOP1MT
releases the tension generated in the circular mitochondrial genome during replication and transcription.
3.2. Proteins involved in mtDNA replication
Genetic information is contained in both strands
of mammalian mtDNA (Fig. 1). Transcription initiates at two promoters in the NCR, one for each
strand, hence heavy strand promoter (HSP), and light
strand promoter (LSP). These give rise to polycistronic transcripts. H-strand transcription starts at
nucleotide position 561 located within HSP, whereas
L-strand transcription starts at nucleotide position
407 within LSP. The initiation of transcription is
facilitated by mitochondrial transcription factor A
(TFAM),(30) and mitochondrial transcription factor B
(TFBM).(31) RNA synthesis in mitochondria is mediated by MTRPOL, in which the conserved C-domain
specifies promoter selectivity and polymerase activity.(32) A second HSP is located immediately outside
the NCR within the tRNAPhe gene. A ‘transcription
termination factor’ (mTERF) simultaneously binds
to this site and another position immediately downstream of the two rRNA genes, thereby forming a
DNA loop, and then generating truncated H-strand
The machinery of mtDNA replication in mammals involves a host of proteins. DNA polymerase γ,
POLG, is believed to be the replicative polymerase
for mtDNA. It forms a heterotrimer(20) composed of
one molecule of POLG1, which is the catalytic subunit, and two molecules of POLG2, which are accessory subunits required for synthesizing long stretches
of DNA in vitro.(21) The catalytic subunit, POLG1,
has one conserved domain for polymerase activity
located towards the C-terminus; at the other end is a
domain for 3’ 5’ exonuclease ‘proofreading’ activity.(22)
Mitochondrial single-stranded DNA-binding
protein (mtSSB) has an N-terminal domain similar in
sequence to prokaryotic SSB.(23) Like its counterparts
elsewhere, it binds single-stranded DNA cooperatively as tetramers. (24) In flies and frogs, mtSSB
enhances the functions of POLG in various assays of
primer-template binding, 3’ 5’ exonuclease activi-
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4. Transcription, RNA processing, post-transcriptional modification, and translation
4.1. Transcription of human mtDNA and processing
of primary transcripts
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transcripts containing the rRNAs with little else.(33)
In the absence of mTERF, full-length H-strand transcripts are produced, which specify 10 mRNAs and
14 tRNAs. Since tRNA genes abut mRNAs in the
polycistronic transcripts, cleavage of tRNAs by
RNase P and RNase Z yields mature mRNAs. (34)
Thus there is no requirement for a complex spliceosome in mitochondria.
4.2. Post-transcriptional modifications
Post-transcriptional modifications of several
bases in excised tRNAs are important for folding,
aminoacylation, and codon recognition. Maturation
of tRNAs requires addition of CCA to the 3’ end for
the attachment of amino acids. The 3’ ends of
mRNA undergo polyadenylation, which is believed
to stabilize transcripts.
4.3. Translation
Mitochondrial ribosomes are broadly prokaryotic-like. They are sensitive to aminoglycosides and
chloramphenicol, and resistant to cyclohexamide(35)
Mammalian mitochondrial ribosomes contain relatively little RNA, but have a high protein content of
29 and 48 polypeptides in the small and large ribosomal subunits, respectively. The result is a low sedimentation coefficient of 55S, compared with the 80S
of nuclear ribosomes and 70S of bacterial ribosomes.
Mitochondrial mRNAs have neither upstream
untranslated regions to facilitate ribosomal binding,
nor a cap structure at the 5’-end to direct ribosomes
to the initiation codon. Instead, the small subunit of
mitochondrial ribosomes binds mRNA in a
sequence-independent way, and moves to the initiation codon. There the small subunit is bound by
mitochondrial translation initiation factor 2 (mtIF2)
and N-formylmethionyl-tRNA, facilitating recruitment of the large ribosomal subunit. Elongation of
translation is carried out by mitochondrial elongation
factors (mtEFs) that are homologous to EF-Tu, EFTs, and EF-G of E. coli. Likewise, termination of
translation is believed to involve the recognition of
stop codons by mitochondrial release factors
(mtRFs) homologous to those in E. coli.
5. Nucleoids
In vivo DNA is rarely, if ever, naked. Rather, it
adopts an ordered condensed structure in association
with a number of proteins. In bacteria, such nucleic
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acid-protein complexes are termed nucleoids. (36)
Packaging of DNA with protein offers a means of
protection from extraneous damage, facilitating
orchestrated copying and segregation of DNA molecules. Both are processes essential to the faithful
transmission of genetic material.
Mitochondrial DNA is no exception; it also
forms nucleoprotein complexes, which have become
the subject of intense study in recent years. (37)
Mitochondrial nucleoprotein complexes, or mitochondrial nucleoids (mt-nucleoids), are believed to
contain multi-copy mitochondrial genomes based on
microscopic studies in which the number of mtDNA
foci in a cell, indicated by a fluorescent dye specific
to DNA, was significantly less than the mtDNA copy
number. In humans, mt-nucleoids are about 0.068
µm in diameter as judged from immunocytochemical
staining, and the mitochondrial genome per nucleoid
is estimated to be between 2 and 10 copies.(38,39) In S
cerevisiae, mt-nucleoids are larger than humans. In
yeast growing aerobically, mt-nucleoids are 0.2 to
0.4 µm in diameter, and the mitochondrial genome
per nucleoid is estimated to be 1 to 2 copies. Under
anaerobic conditions, yeast mt-nucleoids grow to 0.6
to 0.9 µm and each contains the equivalent of ~20
copies of the mitochondrial genome.(37)
Most progress in understanding mitochondrial
nucleoids has been made in yeast. The concept of
mitochondrial nucleoids is based on in vivo and ex
vivo observations that yeast mtDNA forms stable
complexes which are the units of segregation within
the mitochondrial network. Yeast studies have led the
field, largely because yeast genetics allows the rapid
creation and screening of mutants. Caron et al.(40) first
identified a histone-like protein from yeast mitochondria. This protein is encoded by the ABF2 gene.
It has two high-mobility-group (HMG) domains
capable of binding DNA and is required for maintenance of mtDNA.(41) The bacterial DNA packaging
protein HU can restore mtDNA stability to abf2
mutant cells when targeted to mitochondria.(42) Thus
Abf2 is likely to function, at least in part, as a DNApackaging protein. However, it is not clearly understood how Abf2 is involved in the maintenance of
the mitochondrial genome because Yhm2, which is
an inner membrane protein with in vitro DNA binding activity, and aconitase (Aco1), which is required
for TCA cycle, can both complement abf2 mutant
cells.(43,44)
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In organello formaldehyde cross-linking experiments identified a number of candidate mitochondrial nucleoid proteins of yeast;(45) subsequently several
of these have been shown to associate closely with
mtDNA and to contribute to mtDNA stability.(44,46,47)
These include Abf2, Aco1, Rim1, Mgm101, Hsp60,
and Ilv5. Rim1 is a single-stranded binding protein
and by analogy with other systems is presumed to be
involved in yeast mtDNA replication.(48) Mgm101
binds DNA and is required for the repair of damaged
mtDNA. (49) Further investigation revealed that in
vivo, Mgm101 colocalized with actively replicating
mtDNA.(50) Even in the absence of mtDNA, Mgm101
forms a self-replicating complex with an outer membrane protein Mmm1, which itself is required for
mtDNA stability.(51) Hsp60, a well known mitochondrial chaperone and analogue of E. coli GroEL, was
found in yeast mitochondrial nucleoprotein preparations; when tested in vitro it was shown to bind to
mtDNA ‘ori-like sequences’ preferentially. (45)
Moreover, mtDNA is unstable in a hsp60 mutant and
further studies showed that this instability was
caused by a defect in mtDNA transmission to daughter cells,(47) suggesting the nucleoid dynamics underlying mtDNA transmission are regulated by the
interaction between Hsp60 and mtDNA ori
sequences. Ilv5 participates in branched-chain amino
acid biosynthesis and plays a direct role in the parsing of mtDNA into nucleoids.(46) Ilv5 is essentially
two proteins glued together, as the two functions are
quite separable.(52) These findings provide convincing
evidence of highly organized mitochondrial
nucleoids whose maintenance and transmission is
presumably dependent on mechanisms similar to
bacteria. However, yeast mitochondria have few
obvious direct homologues of bacterial DNA maintenance and segregation proteins.(36,37)
Few protein components of yeast mitochondrial
nucleoids have sequence-conserved homologues in
mammals. In addition, targeted mutation of mammalian cells is cumbersome and laborious.
Therefore less is known about mammalian mitochondrial nucleoids; they contain Twinkle, a DNA helicase, which is localized with mtDNA when overexpressed in cultured cells,(26) and TFAM,(53) which is
believed to be the major packaging protein of mammalian mtDNA.(54) TFAM is a homologue of yeast
Abf2, based on the conserved high-mobility-group
(HMG) domains and its complementary effect in
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yeast cells devoid of Abf2. (55) Both Twinkle and
TFAM are essential for mtDNA maintenance.(56,57) A
fraction of POLG and mtSSB colocalize with
Twinkle and mtDNA, as would be expected. (58)
Additional proteins co-purify with frog oocyte
mtDNA, (59) although the role of these, if any, in
mtDNA metabolism is unknown.
Studies of yeast mitochondrial nucleoid dynamics have been aided by live-staining with 4’, 6diamidino-2-phenylindole (DAPI). DAPI is a fluorescent dye that can pass through intact membranes
to bind mtDNA and nuclear DNA in yeast.
Unfortunately DAPI stains human mtDNA weakly.
Recently PicoGreen, a fluorescent dye that binds
specifically to double strand DNA in solution, has
been used to stain mtDNA in living mammalian cells
with good results.(60)
6. Recent progress in mammalian mitochondrial nucleoids
A new procedure using the bacterial nucleoid
protein HU to capture mitochondrial nucleoprotein
from rat liver identified ATAD3, a member of
the AAA+ family of ATPases.(61) Repressing expression of the homologue of ATAD3 in C. elegans
(F54B3.3) caused embryonic lethality, larval arrest,
slow growth, and maternal and progeny
sterility, indicating it is an important mitochondrial
protein involved in cell viability (http://www.wormbase.org/db/gene/gene?name=F54B3.3). Other members of the AAA+ family(62) include the bacterial
nucleoid protein FtsK,(63-65) eukaryotic initiation protein Cdc6, and components of the origin recognition
complex (Orcs 1, 4 and 5) required for the initiation
of DNA replication.(66)
ATAD3 was studied in further detail using RNA
interference in human 143B cells.(61) Dramatic reductions in the PicoGreen signal of mt-nucleoids were
revealed in ATAD3 siRNA-treated cells, suggesting
that it plays a role in mtDNA metabolism (Fig. 2).
In ATAD3 gene-silenced cells, DNA analysis by
neutral/neutral two-dimensional agarose gel electrophoresis indicated that ATAD3 binds within the
NCR of human mtDNA and contributes to the maintenance or formation of mtDNA multimers. (61,67)
Hence, a coherent hypothesis is beginning to emerge.
ATAD3 binds specifically to a region of mtDNA in
the vicinity of the NCR, possibly the D-loop itself,
and this brings together or stabilizes multiple
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PicoGreen
A
Mitotracker
360
Merged
Zoom x 4
Control
Zoom x 2
Zoom x 1
PicoGreen
B
Mitotracker
Merged
Zoom x 4
siRNA
Zoom x 2
Zoom x 1
Fig. 2 The PicoGreen signal of mitochondrial nucleoids is greatly reduced after 2 rounds of ATAD3-siRNA. Panel A, human 143B
cells mock transfected with nuclease free water (Ambion, Huntingdon, U.K.) were live-stained with PicoGreen (Invitrogen, Paisley,
U.K.; green) and Mitotracker (Invitrogen, Paisley, U.K; red in the panel). Panel B, human 143B cells with siRNA ATAD3 treatment
were stained with PicoGreen (green) and Mitotracker (red). Magnifications were with a 60x objective lens with zoom x 4, 2, or 1,
using a Radiance 2000 confocal microscopy system (BIORAD, Hemel Hempstead, U.K.).
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mtDNA molecules.
The obvious next step was to investigate the
DNA binding properties of ATAD3. Two fragments
of recombinant ATAD3 for in vitro DNA binding
assays showed that one (residues 44-247) preferentially binds to D-loop structures,(61) even in solutions
where double-stranded DNA is 1,000 times more
abundant. Coupled with the 2D-AGE data, it is
strongly suggested that ATAD3 binds to clusters of
mitochondrial D-loops through the N-terminal onethird of the protein. The three-stranded stretches of
D-loop that frequently turn up in mtDNA might provide a scaffold for a protein that helps mitochondria
organize their DNA.
The N-terminal one-third of the protein
(residues 44-247) had also been used to raise an antibody which was used to examine the distribution of
ATAD3 relative to mtDNA. Confocal microscopy
indicated that ATAD3 is localized exclusively within
mitochondria (Fig. 3A). However, ATAD3 is not distributed uniformly throughout mitochondria but is
restricted to discrete foci (Fig. 3A), a characteristic
A
B
1
2
3
4
5
C
Fig. 3 ATAD3 co-localized with mitochondria and mtDNA. Panel A, localization of mitochondria and ATAD3. Human 143B cells
were live-stained with Mitotracker (red), then fixed, permeabilized, and immuno-labelled with a rabbit primary antibody against
human ATAD3 (a kind gift from MRC Dunn Human Nutrition Unit, Cambridge, U.K.), and then with a goat anti-rabbit IgG secondary antibody pre-labelled with Oregon Green® 488 (Invitrogen, Paisley, U.K.; green). Panel B, localization of ATAD3 and DNA.
Human 143B cells were fixed, permeabilized, and immuno-labelled for ATAD3 (as in panel A; green). In addition the cells were
labelled with a mouse monoclonal anti-DNA IgM primary antibody (PROGEN Biotechnik, Heidelberg, Germany), and a goat antimouse secondary antibody Cy3 (Amersham Bioscience, Little Chalfont, U.K.; red). Panel C, enlargement of selected regions from
the merged image of panel B. Cells were examined by confocal microscopy using an Olympus FluoView 1000 confocal microscopy
system (Olympus, KeyMed House, U.K.), with magnifications from a 60x objective lens and zoom x 4 in panel A, and a 60x objective lens with zoom x 6 in panel B, in conjunction with the computer program Photoshop Elements (Adobe Systems Inc., Uxbridge,
U.K.) in panel C.
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Mitochondrial DNA instability
of mt-nucleoid.(26,61,67) Confocal microscopy further
revealed frequent co-localization (yellow in Fig. 3C1, 3C-2, 3C-3) of ATAD3 (green in Fig. 3B, 3C) and
mitochondrial nucleoids (red in Fig. 3B, 3C).
Nevertheless a fraction of ATAD3 is in the form of
‘filamentous’ strings, flanked by mitochondrial
nucleoids (Fig. 3C-4, 3C-5). Therefore, a model of
mitochondrial nucleoid division has been proposed.(67) This model presumes that ATAD3 binding
to mitochondrial D-loops enhances protein-protein
interactions, thereby creating a filament, which drives apart the mitochondrial nucleoid. This also
implies that mitochondrial nucleoids lacking ATAD3
are incapable of undergoing nucleoid division.
Substantiating this model will require extensive
investigation of cell biology and further in vitro
analysis of ATAD3.
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mtDNA deletions are invariably found in skeletal
muscle but are often absent from blood. The clinical
outcome is highly variable; the onset can range from
birth to late adulthood (> 60 years). The reasons for
this are not fully understood, but it relates in part to
the mutant load in various tissues. The mildest clinical manifestation is progressive external ophthalmoplegia (PEO), which is characterised by droopy eyelids, and restricted eye movement, and may be
accompanied by exercise intolerance. Much more
severe is Kearns-Sayre syndrome (KSS), which
involves cerebellar or cardiac dysfunction. Pearson’s
syndrome (PS) is a fatal infantile disorder which is
characterised by anemia and impaired synthesis of
digestive enzymes in the pancreas. One third of
PEO/KSS/PS patients have the so-called common
deletion that removes 4977 bp of mtDNA
(nucleotide position 8469 to 13447).(68,69)
7. Human mitochondrial diseases
Mitochondrial diseases are a diverse group of
human diseases characterised by defects in any
aspect of mitochondrial function. Often for simplicity, defects relating to energy production, especially
OXPHOS, are considered separately. Since the components of OXPHOS are encoded by mtDNA and
nuclear DNA, and mtDNA is itself dependent on
nuclear genes for its maintenance and expression,
mitochondrial diseases of OXPHOS fall into three
categories, mtDNA mutations as the primary cause,
nuclear DNA defects affecting OXPHOS but unrelated to mtDNA, and nuclear DNA defects as a cause of
mtDNA perturbation.
8. Mitochondrial DNA as the primary cause
Mitochondrial DNA mutations can themselves
be divided into two broad categories, those that
affect translation, and those affecting a specific protein-encoding gene. The 22 tRNA and 2 rRNA genes
are all required for mitochondrial translation; hence,
deleterious mutations in any one will affect the production of all 13 mitochondrially-encoded components of OXPHOS.
8.1. Large-scale mtDNA deletions
The occurrences of a variety of mtDNA deletions (∆-mtDNA, ~1 to 8 kb in size) are mostly sporadic, always heteroplasmic, and always result in the
loss of one or more tRNA genes, therefore affecting
translation of all 13 mitochondrial genes. Such
8.2. Point mutations affecting tRNA or rRNA
The most common pathological mtDNA mutations are located in tRNA genes; somewhat unexpectedly they produce distinct clinical syndromes.
MELAS syndrome (mitochondrial encephalopathy,
lactic acidosis, and stroke-like episodes) usually presents before the age of 40, after normal early development. The majority of MELAS patients have an A
to G transition at nucleotide position 3243 (A3243G)
within the tRNALeuUUR gene,(70) which intriguingly is
within the mTERF binding site (section 4.1).
Another point mutation, A8344G, within the tRNALys
gene, is associated with MERRF syndrome
(myoclonus epilepsy and ragged-red fibres). (71,72)
Ragged-red fibres (RRF) are a key indicator of mitochondrial dysfunction in skeletal muscle; high concentrations of mitochondria stain reddish-purple with
the Gomori Trichrome stain, hence ragged red fibres
mark areas of mitochondrial proliferation. In situ
hybridisation studies indicate that the mitochondria
in ragged red regions contain high levels of mutant
mtDNA. (73) Hence, proliferation appears to be an
attempt to boost mitochondrial output, although this
is ultimately futile in the case of mutant mtDNA, as
RRF are almost invariably cytochrome c oxidase
negative. Both the MELAS and MERFF mutant
tRNAs are non-functional, as they lack the usual
post-transcriptional modification of uridine at the
first position of the anticodon.(74)
The best-known rRNA mutation occurs at
Chang Gung Med J Vol. 32 No. 4
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363
Chih-Chieh Mao, et al
Mitochondrial DNA instability
nucleotide position 1555 in the 12S rRNA.(75) This
A1555G mutation is associated with aminoglycoside
antibiotic-induced deafness, and maternally inherited
non-syndromic deafness. It has been demonstrated
that the new G-C pair, created by the mutation, facilitates the binding of aminoglycoside antibiotics to
12S rRNA,(76) which explains the molecular pathophysiology of aminoglycoside antibiotic-induced
deafness.
8.3. Point mutations affecting protein-coding genes
Point mutations T8993G/C and T9176G/C in
human mtDNA convert a conserved leucine to an
arginine or proline, near the C-terminus of subunit 6
of ATP synthase.(77) Disease severity is directly related to the percentage of mutant mtDNA in the blood.
When the percentage of T8993G mtDNA is less than
70%, patients are asymptomatic or oligosymptomatic
and when it exceeds the threshold of 70%, NARP
(neurogenic muscle weakness, ataxia, and retinitis
pigmentosa) develops. Point mutations of mitochondrial genes encoding subunits of complex I are associated with LHON (Leber ’s hereditary optic neuropathy) syndrome, a form of adult onset blindness.
The pathogenic mutations are G11778A in ND4,
G3460A in ND1, and T14884C in ND6.
Primary mtDNA mutations can be sporadic, particularly in the case of large deletions. Where mutations are transmitted, they are invariably through the
maternal line. Genetic counselling in the case of
mtDNA mutations is a primitive art as penetrance
and clinical phenotypes are so variable. However in
the case of affected fathers, (almost) complete reassurance can be offered; they will not transmit the disease to their offspring.
9. Nuclear DNA defects affecting OXPHOS
rather than mtDNA
Most of the 1000 or more mitochondrial proteins encoded in nuclear DNA do not directly affect
mtDNA stability, but relate to mitochondrial metabolism and OXPHOS.(78,79) However, since OXPHOS is
formed through the complementation of nuclear and
mitochondrial DNA, mutations in nuclear genes
encoding either structural components of
OXPHOS,(80,81) or factors involved in the assembly of
OXPHOS,(82) can all lead to clinical phenotypes similar to mtDNA mutations.
Chang Gung Med J Vol. 32 No. 4
July-August 2009
10.Nuclear DNA defects as a cause of mtDNA
perturbations
Nuclear genes encode all the mtDNA binding
proteins. Some are essential for replication and others are presumed to play roles in maintenance and
segregation. As mitochondria are the major site of
free radical production, protection from oxidative
stress is another key function of the mtDNA binding
proteins. Mutations in these nuclear genes can therefore produce mutations in mtDNA, but unlike primary mtDNA mutations, these are heterogeneous (multiple deletions and/or point mutations). Predictably,
the clinical phenotypes associated with nuclear gene
defects of proteins involved in mtDNA metabolism
are often similar to those linked to specific mtDNA
mutations, but the transmission follows Mendelian
rules rather than a maternal pattern of inheritance.
10.1. POLG1, Twinkle, and Adenine Nucleotide
Translocator 1 (ANT1)
Autosomal dominant progressive external ophthalmoplegia (adPEO) is characterised by multiple
large deletions of mtDNA in not only the ocular and
skeletal muscles, but also the brain and heart.(83-85)
adPEO is genetically heterogeneous with disease
loci mapped to chromosome 15q25, 10q24, and
4q34-35.
Chromosome 15-linked adPEO is caused by
mutations in the POLG1 gene.(86) In fact, mutations of
POLG1 are the most frequent cause of adPEO,
accounting for approximately half of family
cohorts. (86,87) Amino acid position 955 of POLG1,
which is located within the catalytic polymerase
domain, or C-domain, is the most frequent mutation
site. (87) Mutations within the 3’ 5’ exonuclease
domain, or N-domain, are associated with a recessively inherited form of PEO. Mutations of POLG1
are also associated with Parkinsonism.(88)
Chromosome 10-linked adPEO is caused by
mutations in Twinkle, a DNA helicase, which colocalizes with mtDNA(26) and is a component of mitochondrial nucleoids.(57) Mutations in Twinkle include
various missense mutations and an in-frame duplication of 13 amino acids (dup352-364).(26,89) A recent
study showed that transgenic mice expressing mutant
Twinkle with in-frame dup352-364 develop multiple
mtDNA deletions and a late-onset mitochondrial disease.(90)
Chromosome 4-linked adPEO is caused by
Chih-Chieh Mao, et al
Mitochondrial DNA instability
mutations in adenine nucleotide translocator 1
(ANT1).(91) The familial mutation substitutes a proline for a highly conserved alanine at position 114 in
the ANT1 protein. ANT1 is a heart- and muscle-specific form of the ADP/ATP transporter, which is
located in the mitochondrial inner membrane and is
responsible for transporting ADP in and ATP out of
mitochondria. It is likely that the mutation disturbs
mitochondrial nucleotide pools, although it is not
clear why this should cause multiple mtDNA deletions rather than depletion (as in the next section).
10.2. Thymidine Kinase 2 (TK2) and Deoxyguanosine
Kinase (dGK)
Mutations in the genes encoding thymidine
kinase 2 (TK2) (92) and deoxyguanosine kinase
(dGK)(93) are associated with mtDNA depletion syndrome (MDS), a severe infantile disease inherited in
an autosomal recessive pattern. Patients develop tissue-specific depletion of mtDNA, with its level in
the liver dropping to less than 10% of normal.(94,95) In
mitochondria, both TK2 and dGK are required for
the synthesis of deoxyribonucleoside triphosphates
(dNTPs), the building blocks of DNA. In differentiated neurons and muscle cells, nuclear DNA replication is suspended and the cytosolic dNTP pool is
lowered accordingly. There is no known de novo
synthesis of dNTP in mitochondria, and so deoxyribonucleosides have to be recycled to replenish the
mitochondrial dNTP pool. The salvage pathway for
pyrimidines initially generates dTMP and dCMP by
the action of TK2, whereas dGK generates dAMP
and dGMP for purine biosynthesis. There is evidence
showing that dNTPs need to be in balance for optimal DNA synthesis;(96) an excess or deficiency in any
one dNTP can result in error-prone or complete cessation of DNA synthesis.(97,98)
10.3. Thymidine Phosphorylase (TP)
Mutations in the gene encoding thymidine phosphorylase (TP) (99) are associated with depletion
and/or multiple deletions of mtDNA in the muscles
of patients with MNGIE (Mitochondrial neurogastrointestinal encephalomyopathy), an autosomal
recessive disease. Intriguingly TP is not located in
mitochondria, but in the cytosol where it is involved
in the salvage of thymidine by catalysing the conversion of thymidine to phosphoribose and thymine.
Mutations of TP lead to a decrease in enzyme activi-
364
ty to 5% of that in controls,(99) which results in accumulation of thymidine and an imbalance of dNTP
pools.(100)
10.4. TFAM
TFAM is a component of mitochondrial
nucleoids.(54,55) Although TFAM has no known genetic relationship with any mitochondrial disease, in
mice TFAM knockout causes severe mtDNA depletion and death in the fetus.(56) Tissue-specific disruption of TFAM causes dilated cardiomyopathy, anaesthesia-related conduction block in the heart,(101) and
impaired insulin secretion in pancreatic-β-cells.(102)
11. The importance of dissecting the mitochondrial nucleoid in the pathogenesis of mitochondrial DNA instability
Since there are more than 1000 copies of
mtDNA in each cell, the percentage of mutant
mtDNA needs to exceed a threshold to cause cell
dysfunction. The threshold varies according to energy requirements, with the brain, heart, and skeletal
muscle being the most susceptible to mitochondrial
dysfunction. Crossing the threshold may result from
random genetic drift(103) or biased segregation.(104,105)
From studies on cultured cells it is known that different nuclear backgrounds confer selective advantages
to particular mitochondrial genotypes. (105,106)
Identifying and characterizing all the nucleus-encoded proteins that are functionally associated with
mitochondrial nucleoids will help elucidate the production or biased segregation of mutant mtDNA.
These studies are relevant to patients as the same
mechanisms are likely to be key determinants of
mutant load in particular individuals and tissues.
12. Summary
This review reports the basic facts about mitochondria and mtDNA, and introduces nucleus-encoded proteins that are involved in the metabolism of
mtDNA, with special emphasis on POLG, Twinkle,
and TFAM. Integrated knowledge about the protein
components of the genetic units of mtDNA, namely
mitochondrial nucleoids, is also included. Among
them Twinkle, TFAM, and the recently identified
ATAD3 are detailed. Based on the molecular causes
of mtDNA instability, mitochondria DNA diseases
are grouped into two categories, mtDNA as the primary cause, and nuclear DNA defects as a cause of
Chang Gung Med J Vol. 32 No. 4
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365
Chih-Chieh Mao, et al
Mitochondrial DNA instability
mtDNA perturbations. However, key questions
remain to be answered, in particular the production
and biased segregation of mtDNA instability in specific tissues. These fields may be greatly advanced
by further identification and investigation of the individual components of mitochondrial nucleoids.
Acknowledgements
We acknowledge Chang Gung Memorial
Hospital (Taoyuan, Taiwan), especially Professors
Ping-Wing Lui, Min-Wen Yang, and Angie C-Y Ho
of the Department of Anesthesiology, for their
encouragement and support. Chih-Chieh Mao is supported by CMRP 1331 from the Chang Gung
Memorial Hospital. Ian J. Holt is supported by the
UK Medical Research Council and the European
Union FP6 Mitocombat Integrated Programme.
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Chang Gung Med J Vol. 32 No. 4
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DNA
Ian J. Holt1
DNA
DNA
DNA
DNA
DNA
DNA polymerase γ (POLG) DNA helicase Twinkle (Twinkle)
transcription factor A (TFAM)
Twinkle TFAM
ATAD3
DNA
DNA
DNA
DNA
POLG1 Twinkle ANT1 TK2 dGK TP
DNA
DNA
2009;32:354-69)
mitochondrial
DNA
(
DNA
Medical Research Council Dunn Human Nutrition Unit, Cambridge, U.K.
97 12 26
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Tel.: (03)3281200 2324;
Fax: (03)3281200 2787; E-mail: [email protected]
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