Human Molecular Genetics, 2017, Vol. 26, No.

13 2515–2525

doi: 10.1093/hmg/ddx146
Advance Access Publication Date: 17 April 2017
Original Article

ORIGINAL ARTICLE

A multi-systemic mitochondrial disorder due to a

dominant p.Y955H disease variant in DNA
polymerase gamma
Triinu Siibak1,†,‡, Paula Clemente2,3,‡, Ana Bratic4, Helene Bruhn3,5,
Timo E.S. Kauppila4, Bertil Macao1, Florian A. Schober2,3, Nicole Lesko3,5,
Rolf Wibom3,5, Karin Naess3,5, Inger Nennesmo6, Anna Wedell2,5,7,
Bradley Peter1, Christoph Freyer2,3,5, Maria Falkenberg1,* and
Anna Wredenberg2,3,5,*
1
Department of Medical Biochemistry and Cell Biology, Institute of Biomedicine, University of Gothenburg,
Gothenburg SE-405 30, Sweden, 2Max Planck Institute Biology of Ageing - Karolinska Institutet Laboratory,
Division of Metabolic Diseases, Department of Laboratory Medicine, Karolinska Institutet, Stockholm, SE-171
77, Sweden, 3Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm SE-171 77,
Sweden, 4Department of Mitochondrial Biology, Max Planck Institute for Biology of Ageing, Cologne D-50931,
Germany, 5Centre for Inherited Metabolic Diseases, Karolinska University Hospital, Stockholm SE-171 76,
Sweden, 6Department of Pathology, Karolinska University Hospital, SE-171 77 Stockholm, Sweden and
7
Department of Molecular Medicine and Surgery, Karolinska Institutet, Stockholm SE-171 76, Sweden
*To whom correspondence should be addressed. Tel.: þ46 31 7863444; Email: [email protected] (M.F.); Tel.: þ46 8 51771425;
Email: [email protected] (A.W.)

Abstract
Mutations in the mitochondrial DNA polymerase, POLG, are associated with a variety of clinical presentations, ranging from
early onset fatal brain disease in Alpers syndrome to chronic progressive external ophthalmoplegia. The majority of
mutations are linked with disturbances of mitochondrial DNA (mtDNA) integrity and maintenance. On a molecular level,
depending on their location within the enzyme, mutations either lead to mtDNA depletion or the accumulation of multiple
mtDNA deletions, and in some cases these molecular changes can be correlated to the clinical presentation. We identified a
patient with a dominant p.Y955H mutation in POLG, presenting with a severe, early-onset multi-systemic mitochondrial dis-
ease with bilateral sensorineural hearing loss, cataract, myopathy, and liver failure. Using a combination of disease models of
Drosophila melanogaster and in vitro biochemistry analysis, we compare the molecular consequences of the p.Y955H mutation
to the well-documented p.Y955C mutation. We demonstrate that both mutations affect mtDNA replication and display a
dominant negative effect, with the p.Y955H allele resulting in a more severe polymerase dysfunction.

†
Present address: Cologne Excellence Cluster on Cellular Stress Responses in Ageing-Associated Diseases (CECAD), University of Cologne, 50931 Cologne, Germany.
‡
These authors equally contributed to this work.
Received: February 24, 2017. Revised: April 5, 2017. Accepted: April 11, 2017
C The Author 2017. Published by Oxford University Press.
V
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/
licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited.
For commercial re-use, please contact [email protected]

2515
 2516 | Human Molecular Genetics, 2017, Vol. 26, No. 13

Introduction Results
The mitochondrial DNA polymerase c (POLc) is required for rep- Subjects
lication of the mitochondrial genome (mtDNA). The holoen-
Subject 1 presented with progressive PEO and ovarian failure with
zyme consists of the catalytic subunit POLcA, encoded by the
infertility in adulthood. Measurements of mitochondrial OXPHOS
POLG gene (MIM 174763), and by the dimeric processivity factor
function in a skeletal muscle biopsy showed a normal result (Fig.
POLcB, encoded by the POLG2 gene (MIM 604983) (1). POLcA be-
1A and B), although morphological analysis revealed a high num-
longs to the family A polymerases, with an N-terminal 30 –50 exo-
ber of COX negative muscle fibres (Fig. 1C), as well as paracrystal-
nuclease domain, a central linker domain and a C-terminal
line inclusions by electron microscopy (Supplementary Material,
polymerase domain (2). Replication of the mitochondrial ge-
Fig. S1A), indicating mitochondrial dysfunction.
nome is independent of the cell cycle with individual mtDNA
Subject 2 presented with a slowly progressive multi-
molecules being randomly selected for replication, a phenome-
systemic disorder at the age of 8 months, with weight loss, bilat-
non referred to as relaxed replication (3–6). The total mtDNA
eral sensorineural hearing loss, bilateral cataract, myopathy,
copy number, however, is maintained at a relatively constant
liver failure and feeding difficulties that required a percutane-
level.
ous endoscopic gastrostomy. He had a normal intellectual ca-
Defects in mtDNA replication or nucleotide metabolism
pacity and development. Measurements of mitochondrial
can lead to rearrangements, deletions, point mutations, or de-
OXPHOS activity in a skeletal muscle biopsy was unremarkable
pletion of mtDNA, often resulting in mitochondrial dysfunc-
at 10 months of age, but a reduced ATP production rate was de-
tion and ultimately mitochondrial disease (7). Although
termined in a second biopsy around 1 year later (Fig. 1D and E).
several factors involved in mtDNA replication have been asso-
Morphological analysis confirmed a mitochondrial dysfunction,
ciated with mitochondrial diseases, mutations in POLG are
exhibiting a high number of COX negative muscle fibres (Fig. 1F)
most common with close to 230 different disease-causing mu-
and ragged-red fibres (Fig. 1G).
tations reported (8) [recently summarized in (9)]. The associ-
ated clinical symptoms can be quite variable, both with
respect to disease onset and clinical presentation, and cause a The p.Y955H mutation in POLcA is associated with early
number of different disease entities, such as Alpers syndrome
onset multi-systemic mitochondrial disease
(MIM 203700), mitochondrial neurogastrointestinal encepha-
lopathy (MNGIE: MIM 613662), sensory ataxic neuropathy, dys- Sequencing of the POLG locus in subject 1 revealed that the pa-
arthria and ophthalmoparesis (SANDO: MIM 607459), tient was heterozygous for the previously reported c.2864A>G,
spinocerebellar ataxia-epilepsy (SCAE: MIM 607459), and p.Y955C, mutation (13) (Fig. 1H). Southern blot analysis showed
chronic progressive external ophthalmoplegia (CPEO: MIM the accumulation of multiple mtDNA deletions (Supplementary
157640 and MIM 258450). On a molecular level, mutations in Material, Fig. S1B), while mtDNA sequencing was unremarkable.
POLG lead to the accumulation of multiple mtDNA deletions or Sequencing of POLG in subject 2 detected a previously unre-
mtDNA depletion (5,9), which in turn cause reduced oxidative ported mutation, c.2863T>C, p.Y955H, additionally revealing a
phosphorylation (OXPHOS). heterozygous p.Q1236H mutation, previously shown to be be-
CPEO is the most common mitochondrial myopathy, defined nign (22). Detailed allele-specific analysis revealed that the
by a progressive bilateral ptosis and diffuse, symmetric reduc- p.Y955H mutation was in cis with p.Q1236H on the paternal
tion in ocular motility, often associated with additional symp- POLG allele but absent in the father (Table 1). The p.Y955H mu-
toms, e.g. hearing loss and ataxia [reviewed in (10)]. About half tation had thus occurred de novo. Multiplex ligation-dependent
of all CPEO cases are inherited and disease-causing mutations probe amplification (MLPA) and cDNA analysis of POLG did not
at seven different loci have so far been identified, including loci detect any other disease-causing mutation in the gene. Whole
coding for the mitochondrial adenine nucleotide translocator 1 exome sequencing on DNA samples from subject 2, was per-
(SLC25A4: MIM 103220) (11), the mitochondrial DNA helicase, formed as described previously (23,24), but revealed no other
TWINKLE (TWNK: MIM 606075) (12) and POLcA (13). The p.Y955C potentially disease-causing mutation involved in metabolic or
mutation of human POLG results in adult onset autosomal dom- mitochondrial diseases.
inant CPEO (13) as well as premature ovarian failure (14) and is Previous reports suggested that mutations affecting position
associated with the accumulation of multiple mtDNA deletions Y955 in human POLcA can lead to an increase in incorrect nu-
in affected patients (13,15). Mutagenesis experiments identified cleotide incorporation, but cloning and sequencing of mtDNA
Y955, together with residues R943, L947 and A957 of POLcA to be from human muscle samples showed no difference in point
essential for nucleotide specificity, and processivity (16–21), mutation load, when compared with control samples
with the Y955C mutation resulting in replicative stalling and (Supplementary Material, Table S1).
formation of multiple mtDNA deletions (21).
We here identify an autosomal dominant p.Y955H mutation
Mutations at p.Y873 are recessive lethal in Drosophila
in POLG, leading to a severe multi-systemic mitochondrial dis-
ease with bilateral sensorineural hearing loss, cataract, myopa-
melanogaster
thy and liver failure in a paediatric patient. Using disease In order to understand the molecular and physiological conse-
models of Drosophila melanogaster (Dm) and in vitro characteriza- quences of the p.Y955H mutation we generated Drosophila mela-
tion, we demonstrate that the p.Y955C and p.Y955H mutations nogaster (Dm) models for both the p.Y955C and p.Y955H
both affect mtDNA replication and have a dominant negative mutations (Y955 in human POLcA corresponds to Y873 in
effect on DNA synthesis. The p.Y955H mutation resulted in a DmPOLcA). To this end we targeted the Dm tamas locus, coding
more severe dysfunction than p.Y955C, in agreement with the for DmPOLcA, following a previously described procedure (25)
clinical phenotype of the patient, which is unusually severe for (for details see materials and methods) (Supplementary
a dominant POLG disease. Material, Fig. S2).
 Human Molecular Genetics, 2017, Vol. 26, No. 13 | 2517

Figure 1. Characterization of mitochondrial function in skeletal muscle. (A) Mitochondrial ATP production rate (MAPR) in fresh skeletal muscle mitochondria, isolated
from subject 1, using six different substrate combinations as indicated. Results are presented as the ATP synthesis rate (units) per unit of CS activity (control n¼11; age
0–5 years). (B) Respiratory chain enzyme activities of complexes I and III (NADH:cytochrome c reductase), complex I (NADH:coenzyme Q reductase), complexes II and III
(succinate:cytochrome c reductase, SCR), complex IV (cytochrome c oxidase) and citrate synthase (CS) were determined. Results are presented as percentage of mean
control (n¼9; age 0–5 years) values. The range of control values is depicted as 6 2SD. (C) COX/SDH double staining of fresh-frozen skeletal muscle from subject 1. (D)
MAPR as in (A) from subject 2. (E) Respiratory chain enzyme activities as in (B) from subject 2. (F) COX/SDH double staining of fresh-frozen skeletal muscle from subject
2. (G) Gomori-trichrome staining of fresh-frozen skeletal muscle from subject 2 revealing ragged-red fibres (RRF). (H) Electropherogram of POLcA from subject 1, 2 and
control.

Table 1. Nucleotide and amino acid changes in POLG in subject 2 whereas Dm lines homozygous for either of the two mutations
and his parents were larval lethal at the third instar larval stage (Fig. 2D).

Nucleotide Protein

Subject 2 c.[2863T>C; 3708G>T];[¼] p.[Y955H; Q1236H];[¼] The p.Y873H and p.Y873C mutations lead to mtDNA
Father c.[3708G>T];[¼] p.[Q1236H];[¼] depletion in Drosophila melanogaster
Mother c.[¼];[¼] p.[¼];[¼]
The p.Y955C mutation causes multiple mtDNA deletions in hu-
mans (13,15) as observed in muscle samples from subject 1
(Supplementary Material, Fig. S1B). In contrast, no multiple de-
letions were observed in muscle samples from subject 2, carry-
Flies heterozygous for the p.Y873C and p.Y873H mutations ing the p.Y955H mutation (Supplementary Material, Fig. S1B).
did not show any obvious phenotypic abnormalities. Eclosure Interestingly, flies carrying the p.Y873H or p.Y873C mutation
rates were comparable to control flies (Fig. 2A), although flies did not present multiple deletions; neither as heterozygous flies
heterozygous for p.Y873C mutation were developmentally de- (Fig. 3A) nor as homozygous larvae (Fig. 3B). However, qRT-PCR
layed (Fig. 2B). Lifespans (Fig. 2C) were normal even after 15 gen- and Southern blot analysis revealed severe mtDNA depletion in
erations of intercrossing (Supplementary Material, Fig. S3), L3 larvae homozygous for either of the two mutants, with a
 2518 | Human Molecular Genetics, 2017, Vol. 26, No. 13

Figure 2. Eclosion rates and life-spans in flies carrying the p.Y873C or p.Y873H mutations. (A) Eclosure rates, (B) life-span, (C) developmental time of F0 and F1 flies. (D)
L3 larvae of control (wt/wt) and homozygous mutant (Y873C/Y873C and Y873H/Y873H) larvae at 4 days after egg laying.

milder reduction in the heterozygous state (Fig. 3C and D). experiments, using two different primed DNA templates (Fig.
Heterozygous flies (p.Y873H or p.Y873C) showed an mtDNA de- 4B). In the absence of dNTPs, WT POLcA will use its 30 –50 exonu-
pletion, which was somewhat more pronounced after 15 gener- clease activity to digest the labelled primer, but in the presence
ations intercrossing (Fig. 3C). of dNTPs, the polymerase will initiate primer elongation. In this
assay, POLcA:Y955H behaved as previously demonstrated for
POLcA:Y955C and required higher dNTP concentrations to pro-
POLc:Y955H has lower affinity to DNA than WT POLc or duce full-length products (Fig. 4B, upper panel). Increasing the
POLc:Y955C number of thymines on the template strand, promoted stalling
of both mutant polymerases (Fig. 4B, lower panel), consistent
We next investigated the biochemical consequences of the
with preferred stalling at dATP insertion sites (Fig. 4B, compare
p.Y955H mutation and for comparison we also analysed the pre-
upper and lower panel) (21).
viously characterized p.Y955C mutation (20,21). To this end, wild-
type POLcA protein and mutant derivatives (POLcA:Y955C and
POLcA:Y955H) were expressed and purified. DNA binding proper- POLcA:Y955H has a dominant negative effect on the
ties of all three POLcA versions were first assessed in the absence replisome at low dNTP concentrations
or presence of recombinant POLcB, using an electrophoretic mo-
In the presence of mtSSB and the TWINKLE helicase, WT POLcA
bility shift assay (EMSA). Both POLcA:Y955C and POLcA:Y955H
in complex with POLcB is able to replicate a circular dsDNA tem-
bound to a primed DNA template independently of POLcB (Fig.
plate containing a preformed replication fork (Fig. 5A, lanes 1–4
4A). However, the Kd (equilibrium dissociation constant) for bind-
and 13–16) (26). Neither POLcA:Y955H nor POLcA:Y955C could
ing to the template, was higher for POLcA:Y955H than for WT
support DNA synthesis (Fig. 5A, lanes 5–8 and 17–20, respec-
POLcA and POLcA:Y955C (Supplementary Material, Fig. S4).
tively). In agreement with previous reports, POLcA:Y955C dis-
played a dominant negative effect on DNA synthesis in the
presence of WT POLcA (Fig. 5A, lanes 21–24) (21). The dominant
Modifications at position Y955 are associated with
negative effect was less pronounced with POLcA:Y955H even
difficulties of incorporating dATP
when the mutant was added at a 3:1 molar ratio relative to WT
We previously demonstrated that the p.Y955C mutation leads POLcA (compare lanes 9–12 with lanes 21–24 in Fig. 5A).
to stalling at low dNTP concentrations. POLcA:Y955C is espe- Postmitotic tissues contain lower dNTP concentrations than the
cially sensitive to low dATP concentrations, and the enzyme en- 10 mM used here (27,28). We therefore repeated the experiment
ters a polymerase/exonuclease idling mode at dATP insertion using 1 mM dNTP. At this concentration, POLcA:Y955H also dis-
sites (21). To investigate if this is a general phenotype for muta- played a dominant negative effect on WT POLcA activity (Fig.
tions affecting Y955, we performed primer extension 5B, lanes 9–12). Furthermore, we also observed an accumulation
 Human Molecular Genetics, 2017, Vol. 26, No. 13 | 2519

Figure 3. Mutations at p.Y873 lead to mtDNA depletion in flies. (A) Southern blot analysis of mtDNA of heterozygous mutant flies. (B) Long-range PCR of homozygous
mutant and control larvae. Primers were situated as indicated. (C) Southern blot analysis of mtDNA from homozygous mutant larvae. (D) Relative mtDNA levels deter-
mined by qPCR in L3 larvae (L0), or flies intercrossed for 1 (F1) or 15 (F15) generations. TaqMan probes used as indicated. Error bars are 6SD (*P<0.05, **P<0.01, ***P<0.001,
n¼5).

of shorter-than-input length DNA products (Fig. 5B, lane 12), more dNTP, which reasons well with its lower dNTP affinity
suggesting that POLcA:Y955H causes template degradation at (Fig. 6B, lanes 5–7). The POLcA:Y955H mutant on the other hand
lower dNTP concentrations. had problems to fully extend the 60-mer even in the presence of
100 mM dNTP and were therefore unable to create ligateable
ends (Fig. 6B, lanes 8–10). Here again, we observed that both mu-
POLcA:Y955C and POLcA:Y955H display reduced tants have increased exonuclease activity at low dNTP concen-
ligation efficiency trations (see Fig. 6B, lanes 5 and 8). Next, we asked whether the
At the end of replication, DNA ends are ligated to produce a presence of the mutants could affect the ability of the WT to
closed circular mtDNA molecule. We have previously shown create ligateable ends, which would reflect a typical situation
that the 30 –50 exonuclease and 50 –30 polymerase activities of in vivo. To answer this question, we performed time-dependent
POLcA must be correctly balanced in order to support ligation ligation assays at 1 mM dNTPs, where WT POLcA was mixed
(29). Since mutations affecting position Y955 shift the balance with either of the two mutants (Fig. 6C). At this dNTP concentra-
towards the exonuclease activity, we decided to monitor effects tion only the WT protein is able to extend the primer. Addition
on ligation. To this end we performed coupled polymerase- of POLcA:Y955C or POLcA:Y955H severely inhibited polymeriza-
ligation assays, using a template consisting of a radioactively 50 - tion and the ability of WT POLcA to create ligateable nicks (Fig.
labelled 60-mer hybridized to a circular 100-mer (Fig. 6A). On 6C and D).
this template, DNA polymerase needs to extend the 30 end to
full circle and then terminate at the downstream 50 end, in order
to produce a ligateable nick. WT POLcA extended the 60-mer
Discussion
and provided ligateable ends already at 1 mM dNTP (Fig. 6B, Mitochondrial diseases form a highly diverse group, with a
lanes 2–4). The POLcA:Y955C mutant was also capable of gener- range of clinical symptoms and different ages of onset. They are
ating ligateable full-length products, but it needed 100 times predominantly monogenic disorders and recent advances in
 2520 | Human Molecular Genetics, 2017, Vol. 26, No. 13

Figure 4. In vitro DNA binding and polymerization properties of POLcA:Y955C and POLcA:Y955H. (A) Electrophoretic mobility shift assays showing that POLcA:Y955C
and POLcA:Y955H bind DNA both in absence (lanes 5 and 8) and presence (lanes 6 and 9) of POLcB. Lanes 1, 4 and 7 contained no protein. Lower panel: schematic repre-
32
sentation of the DNA template. The asterisk indicates the P label on the 5 end of the 20-mer. (B) Coupled 3’–5’ exonuclease/polymerase assays show that both
POLcA:Y955C and POLcA:Y955H required higher dNTP concentrations than WT POLcA to synthesize DNA (all experiments were performed in the presence of POLcB).
Reactions were run on denaturing 15% PAGE. Below: scheme of the primer-extension reaction starting from the 20-meric primer to produce a 35-mer product.

Figure 5. Rolling circle replication assay reveals dominant negative effect of POLcA:Y955C and POLcA:Y955H. (A) In vitro replication reactions performed at 10 mM dNTP
and indicated POLc versions (all experiments were performed in the presence of POLcB). Lanes 1–4, WT POLcA; lanes 5–8; POLcA:Y955H; lanes 9–12, WT POLcA and
POLcA:Y955H; lanes 13–16, WT POLcA; lanes 17–20; POLcA:Y955C; lanes 21–24, WT and POLcA:Y955C. (B) As in (A) but at 1 mM dNTP.
 Human Molecular Genetics, 2017, Vol. 26, No. 13 | 2521

Figure 6. Effects of POLcA:Y955C and POLcA:Y955H on ligation efficiency. (A) Schematic picture showing the template used in the coupled DNA synthesis-ligation as-
say. (B) Coupled DNA synthesis-ligation assay using different amounts of dNTPs (1, 10, or 100 mM) and indicated POLc versions (all experiments were performed in the
presence of POLcB). Lane l, input template: lanes 2–4, WT POLcA; lanes 5–7, POLcA:Y955C and lanes 8–10 POLcA:Y955H. (C) As in (B) but was performed as a time-course
experiment (5, 15, 30 or 45 min) using 1 mM dNTPs and at a 1:1 ratio of indicated POLc versions. Lanes 1, 6 and 11, input template; lanes 2–5, WT POLcA; lanes 7–10, WT
POLcA and POLcA:Y955C; lanes 12–15, WT POLcA and POLcA:Y955H. (D) Ratio of ligated product compared with total DNA between different mixing-reactions of POLc
versions. Based on quantifications of band-intensity from reactions as shown in (C). Solid black bars are WT POLcA alone, grey bars are WT POLcA mixed with
POLcA:Y955C and white bars represent WT POLcA mixed with POLcA:Y955H. Error-bars are standard deviations from three independent reactions. (E) Molecular model
of Y955 and surrounding amino acids. Upper panel, WT POLcA; lower left panel, POLcA:Y955C; lower right panel, POLcA:Y955H.

sequencing technologies have revolutionized the diagnosis of date, almost 230 mutations have been identified in the human
patients with mitochondrial diseases. Despite these advances, POLG gene alone (8), with both dominant and recessive inheri-
the correlation between genotype and phenotype remains diffi- tance patterns. A substantial number of mutations are also re-
cult to predict and combined with the large heterogeneity of the ported to only prompt clinical symptoms, when inherited in
human genome makes functional validation of novel disease trans with other mutations as compound heterozygous, and it is
variants essential. A complicating factor is that a biochemical thus important to fully characterise variants. We here used a
defect is not always observed in patient samples (30–32). To combination of in vitro biochemistry, model organisms and
 2522 | Human Molecular Genetics, 2017, Vol. 26, No. 13

patient investigations to demonstrate pathogenicity of a muta-

tion in POLG, which causes a tyrosine to histidine change at po-
sition 955 in the human POLcA protein.
The tyrosine residue at position 955 of POLcA is essential for
nucleotide incorporation into the growing DNA strand. An inter-
action between Y955 and E895 is required for the binding of the
incoming nucleotide (Fig. 6E). The hydroxyl group of Y955 inter-
acts with the carboxyl group of E895, which is located adjacent
to the catalytic helix (residues 943–955). In T7 DNA polymerase
this glutamate selectively stabilizes the incoming nucleotide.
Figure 7. A schematic model for the effects of mutant POLcA:Y955C and
The polar contacts, rotomer conformation and steric influence
POLcA:Y955H on replication fork progression. The mitochondrial replication ma-
of E895 likely contribute to the formation of a tight binding chinery at the fork is dynamic and POLc is regularly coming on and off the 3’
pocket. In addition, Y955 may aid catalysis via E895-mediated end of the newly synthesized DNA. When WT POLc (purple) is present at the
alignment of the catalytic helix towards the nucleotide. replication fork of a heterozygous patient, it prevents mutant POLc (orange)
The severity of the p.Y955C and p.Y955H mutations is due, from binding and mtDNA synthesis can therefore progress (left panel). Once
mutant POLc binds the primed template, it enters an idling mode (repeated cy-
in part, to the steric influence of Y955 in the active site (33). The
cles of incorporation and degradation at the primer terminus) and simulta-
large phenyl ring excludes solvent from the active site, while
neously blocks the WT protein from accessing the 3’ end (right panel). mtSSB is
providing a tight binding pocket for the nucleotide. Removal of green and TWINKLE is blue.
this phenyl ring results in a loss of favourable p–p stacking in-
teractions between the incoming base and Y955 (negatively af-
are subtle, they do lead to fundamentally different clinical pre-
fecting nucleotide binding), which likely destabilises the active
sentations. Our data therefore further demonstrates the com-
site. Differences in Kd were also observed between the p.Y955C
plexity of mitochondrial diseases and how difficult it is to
and p.Y955H mutated versions of POLc, with the latter having 5-
predict clinical phenotypes based on molecular defects in these
fold weaker binding to a primed DNA template. This is not sur-
patients.
prising given that the introduction of a polar histidine into an
apolar buried cavity can negatively affect both protein stability
and ligand binding (34–36). Replacement of Y955 with histidine Materials and Methods
is thus likely responsible for (i) loss of steric packing, (ii) loss of a
The Regional Ethics Committee at Karolinska Institutet ap-
stabilizing hydrogen bond with E895, (iii) loss of p-stacking in-
proved the use of patient material in this study.
teractions with Y951 and (iv) electrostatic repulsion within the
binding cavity (Fig. 6E, lower left panel).
The p.Y955C mutation leads to reduced polymerase activity
DNA and RNA isolation from patients
and replication stalling (16–21). Previous data also suggested
that the p.Y955C mutation could cause increased mtDNA muta- Genomic DNA from the patients was isolated from skeletal
tion loads (37–40), but this conclusion was not supported by muscle, blood or fibroblasts, using QIAamp DNA Mini Kit
data obtained here from human muscle. (Qiagen). For RNA analysis, total RNA was isolated from fibro-
The p.Y955C mutation is consistently associated with multi- blasts using RNeasy Mini Kit (Qiagen).
ple deletions in mtDNA, but we could not reproduce this pheno-
type in flies harbouring the p.Y955C mutation. Neither did the
Molecular analysis in patients
p.Y955H mutation lead to multiple deletions in either human
skeletal muscle samples or in the corresponding fly model. The entire mtDNA sequence from the patients was determined
However, this is not surprising, since it has been shown that the from total DNA isolated from skeletal muscle, while the POLG
number of deletion-molecules increases with age. The lack of gene was sequenced from total DNA isolated from the patient’s
deletion in the flies and in the p.Y955H patient could simply re- blood. MtDNA was amplified by PCR in 28 overlapping M13-
flect the short lifespan of flies and the very young age of the pa- tailed fragments as described before (42). The coding exons 2–23
tient. In humans, multiple mtDNA deletions do not become of POLG were amplified by PCR using M13-tagged intronic pri-
visible in Southern blots until 20 years of age (41). mers. Subsequent sequencing of all PCR products was carried
Nevertheless, we observed mtDNA depletion with both p.Y873C out with M13 primers, using the BigDye version 3.1 sequencing
and p.Y873H, consistent with observations made in murine and kit (Applied Biosystems) on a 3130xl Genetic Analyser (Applied
yeast models of the p.Y955C mutation (18,37–40). Depletion is Biosystems). MtDNA sequence data were compared with the re-
also consistent with results from the in vitro characterization of vised Cambridge reference sequence for human mtDNA
the mutants. Both POLcA:Y955C and POLcA:Y955H were unable (GenBank NC_012920.1) and for POLG to the reference sequence
to synthesise DNA and had a dominant negative effect on the NM_002693.2. MLPA analysis, using kit P010 (MRC Holland,
DNA synthesis in the presence of WT POLcA. We favour a model Amsterdam), for detection of deletions or duplications of each
in which the mutant proteins are competing with WT POLcA for exon of the POLG gene was performed according to the manu-
the primer terminus, thereby slowing down replication fork pro- facturer’s protocol as described earlier (43). RT-PCR was per-
gression (Fig. 7). formed on isolated RNA, using GeneAmp RNA PCR kit (Applied
As demonstrated here, the p.Y955C and p.Y955H mutations Biosystems) and exons 2–23 of POLG cDNA were amplified by
cause related, but distinct molecular phenotypes. Whereas PCR in 10 overlapping fragments. The size and amount of the
POLcA:Y955H is unable to synthesise DNA, POLcA:Y955C has PCR products was compared with a control sample in 2% aga-
strongly impaired DNA synthesis activity that can be partly rose gel. Sequencing of the PCR products from cDNA was per-
overcome at high dNTP concentrations. Furthermore, formed as described for genomic DNA above. Southern blot
POLcA:Y955C has a stronger affinity for primed DNA templates, analysis was done using aliquots of 0.2 mg total DNA isolated
compared with POLcA:Y955H. Even if the molecular differences from skeletal muscle, which were digested with the restriction
 Human Molecular Genetics, 2017, Vol. 26, No. 13 | 2523

enzyme PvuII (New England Biolabs) and fractionated by elec- with CircLigase TMssDNA ligase (Epicentre), followed by anneal-
trophoresis in 0.5% agarose gels. The DNA was then transferred ing to a 32P-labelled 60-mer (50 -TCT GGT TAG GCT GGT GTT AGG
to Hybond XL nylon filter (GE Healthcare) by capillary blotting GTT CTT TGT TTT TGG GTT TGG CAG AGA TGT GTT TAA GTG-
under standard procedures. The filter was hybridized with an 30 ). The reactions were performed in a volume of 20 ml contain-
equimolar mix of radiolabelled mtDNA probes corresponding to ing 20 mM Tris–HCl (pH 7.5), 1 mM DTT, 0.1 mg/ml BSA, 10 mM
nucleotides 1–12 640, as described previously (44). MgCl2, 0.5 mM ATP, 4 units of T4 DNA ligase, 10 fmol circular
DNA substrate, indicated POLc version, and varying amounts of
dNTPs as specified in the figure legends. In the mixing experi-
Expression and purification of recombinant human ments, 75 fmol WT POLcA, POLcA:Y955H, or POLcA:Y955C in
proteins complex with 300 fmol POLcB were pre-incubated in the mixture
Mutated versions of POLcA were constructed using the on ice for 10 min before an additional 75 fmol of WT POLcA in
QuikChange Lightning Site-directed mutagenesis kit according complex with 300 fmol POLcB was added to each reaction.
to the provided protocol (Agilent Technologies) and confirmed Reactions were incubated for 30 min if nothing else is indicated
by sequencing (Eurofins MWG Operon). Recombinant baculovi- in the figure legends and terminated with 2 stop buffer
ruses coding for TWINKLE, mitochondrial single-stranded DNA- (Formamide with 10 mM EDTA, 0.025% bromophenol blue,
binding protein (mtSSB), WT POLcA, POLcA:Y955C, 0.025% xylene cyanol). Samples were run in 7 M urea/10% poly-
POLcA:Y955H and POLcB were expressed in Sf9 cells and puri- acrylamide gels and visualized with a PhosphorImager or
fied as previously described (26). autoradiography.

EMSA and Kd determination Drosophila stocks and maintenance

The affinity between POLc (POLcA in complex with POLcB) and a All genomically engineered fly strains were constantly back-
primer template was monitored using an EMSA as previously crossed into a white Dahomey Wolbachia-free (wDahT) WT
described (25). Each experiment was repeated at least three strain. All fly stocks were maintained at 25  C on a 12:12 h light/
times. Band intensities representing unbound and bound DNA dark cycle with 60% humidity and fed on a sugar/yeast/agar
were quantified using Fujifilm Multi Gauge V3.1 software. The (SYA) diet (25).
fraction of DNA bound in each reaction was plotted versus the
concentration of POLc. Data were fitted with the binding equa-
Generation of genomically engineered DmPOLcA flies
tion (Fraction bound ¼ (MaxB  [POLc])/(MaxB þ [POLc]) using
EXCELs Add-in ‘Solver’ to perform non-linear regression and ob- Human POLG sequence (NP_002684.1) was aligned to the
tain values for Kd (as the value corresponding to the midpoint of Drosophila melanogaster homolog (tamas; NP_476821.1) and the
MaxB) and using MaxB set to 1 (the fraction bound at which the fly-equivalent position for p.Y955 was identified at position
data plateaus). p.Y873. Genetically modified flies were generated essentially as
described previously (25,45). We previously cloned the Tamas
locus into the fly-specific targeting vector pGEattB-GMR (25),
Coupled 30 –50 exonuclease/polymerase assays which was here modified by site-directed mutagenesis to intro-
A 20-mer (50 -CGG TCG AGT CTA GAG GAG CC-30 ) labelled at the duce the p.Y873C and p.Y873H mutations. Mutant variants of
50 end with [c-32P] ATP was annealed to a 35-mer oligonucleo- the tamas gene were then injected into the embryos of
tide containing a poly-dT stretch, 50 -TTT TTT TTT TAT CCG GGC DmPolcA (Tamas) KO founder embryos (25), expressing u31 inte-
TCC TCT AGA CTC GAC CG-30 , or an oligonucleotide without a grase by the in-house Drosophila transgenic core facility. Positive
poly-dT stretch (50 -GAC AAC CAG CAG CCG GGC TCC TCT AGA flies were selected by eye colour and confirmed by Southern
CTC GAC CG-30 , as illustrated in Fig. 4B. These two templates blot/sequencing.
were used as substrates to investigate DNA polymerization and
30 –50 exonuclease activity as previously described (21).
Life-span determination
Newly eclosed adult flies were mated for 2 days before they
In vitro rolling circle DNA replication were sorted for lifespan analyses. Two hundred females per ge-
The reaction mixtures (20 ll) contained 10 fmol of rolling circle notype were used at a density of 10 flies per vial. Flies were
template and reactions were performed as described previously transferred to new vials every 2–3 days and dead flies were
except that lower dNTP concentrations (1 mM or 10 mM) were counted.
used. POLc (75 fmol WT POLcA in complex with 300 fmol POLcB,
225 fmol POLcA:Y955C in complex with 675 fmol POLcB, or
Hatching and eclosure rates
225 fmol POLcA:Y955H in complex with 675 fmol POLcB) were
added as indicated in the figure legends (26). Fly development was assessed as follows. Flies were allowed to
lay eggs on grape juice agar plates for 2 h. One hundred eggs per
genotype were individually picked and placed into vials with
Coupled DNA synthesis-ligation assay SYA food. The number of eclosed flies was scored every 12 h. At
The synthesis-ligation assay was adapted from (29). Here a cir- least five biological replicates were done for each genotype.
cular DNA template was used instead of a linear (Fig. 4a). The
closed circular template was constructed by circularizing the
DNA isolation and southern blot analysis from flies
100 nt oligonucleotide (50 -GAG GGG TAT GTG GCC ACA GCA CTT
AAA CAC ATC TCT GCC AAA CCC AAA AAC AAA GAA CCC TAA For relative mtDNA copy number determination, total DNA ex-
CAC CAG CCT AAC CAG ATT TCA AAT TTC ATA CCC CTA T-30 ) tractions were prepared from L3 larvae using DNeasy Blood and
 2524 | Human Molecular Genetics, 2017, Vol. 26, No. 13

Tissue Kit (Qiagen). Five biological replicates, each with 10 lar- and disease-related polymerase mutations. Cell, 139,
vae, were prepared for each genotype. Quantification of mtDNA 312–324.
levels was done using SYBR-Green qPCR analyses and primers 2. Longley, M.J., Nguyen, D., Kunkel, T.A. and Copeland, W.C.
targeting the mitochondrial encoded genes 12S, COX1 and CytB, (2001) The fidelity of human DNA polymerase gamma with
and normalized to the nuclear encoded histone gene. All data and without exonucleolytic proofreading and the p55 acces-
were normalized against wild-type levels. sory subunit. J. Biol. Chem., 276, 38555–38562.
For Southern blot analysis, total DNA was extracted from 20 3. Birky, C.W. (1994) Relaxed and stringent genomes: why cyto-
to 30 L3 larvae, either using the DNeasy Blood and Tissue kit plasmic genes don‘t obey Mendel’s laws. J. Hered., 85,
(Qiagen), or by homogenizing samples with a tissue grinder in 355–365.
400 ml of buffer A (100 mM Tris–HCl, pH 7.5; 100 mM EDTA; 4. Clayton, D.A. (1992) Transcription and replication of animal
100 mM NaCl and 0.5% SDS). After incubation at 65  C for 30 min, mitochondrial DNAs. Int. Rev. Cytol., 141, 217–232.
800 ml of freshly prepared Buffer B (4 ml of 5 M KOAc and 10 ml of 5. Falkenberg, M., Larsson, N.-G. and Gustafsson, C.M. (2007)
6 M lithium chloride) was added and samples were left on ice for DNA replication and transcription in mammalian mitochon-
120 min. After incubation, samples were centrifuged at 12 000g dria. Annu. Rev. Biochem., 76, 679–699.
for 15 min and supernatant was transferred into a new tube. 6. Gustafsson, C.M., Falkenberg, M. and Larsson, N.-G. (2016)
About 540 ml of isopropanol was added to the supernatant and Maintenance and expression of mammalian mitochondrial
samples were further centrifuged at 12 000g for 15 min. Pellets DNA. Annu. Rev. Biochem., 85, 133–160.
were washed with 70% ethanol, dried and resuspended in 100 ml 7. Zeviani, M. and Di Donato, S. (2004) Mitochondrial disorders.
nuclease-free water containing 20 mg/ml RNase A. After incuba- Brain, 127, 2153–2172.
tion at 37  C for 1 h, samples were stored at þ4  C. 8. Human DNA polymerase gamma mutation database human
Approximately 1–3 mg of total DNA was cut using either XhoI or DNA polymerase gamma mutation database. https://tools.
StyI restriction endonuclease (NEB). Digestions were run on 0.8% niehs.nih.gov/polg/.
agarose gels, and blotted to Hybond-N membrane (Amersham 9. Young, M.J. and Copeland, W.C. (2016) Human mitochondrial
Bioscience). COXI or ND6-CytB (positions 10169–11169) were used as DNA replication machinery and disease. Curr. Opin. Genet.
probes, and signals were visualized by autoradiography. 32P-labelling Dev., 38, 52–62.
of Southern probes was done according to manufacturer’s instruc- 10. McClelland, C., Manousakis, G. and Lee, M.S. (2016)
tions (Prime-IT II Random Prime Labelling Kit, Agilent). Progressive external ophthalmoplegia. Curr. Neurol. Neurosci.
Long-Range PCR was performed using primers: Dmel_Long 1: Rep., 16, 53.
5’AAT TAA GCT ACT GGG TTC ATA CCC C3’, Dmel_Long 2: 11. Kaukonen, J., Juselius, J.K., Tiranti, V., Kytta € la
€ , A., Zeviani,
5’GCT CTA AAA TAT GTA CAC ATC GCC C3’, Dmel_Long 3: M., Comi, G.P., Kera € nen, S., Peltonen, L. and Suomalainen, A.
5’TTT GAA GCA GCT GCA TGA TAT TGA C3’ and Dmel_Long 4: (2000) Role of adenine nucleotide translocator 1 in mtDNA
5’AGA GTT TGA CAT TGA AGA TGT TAT GGA G3’. maintenance. Science, 289, 782–785.
12. Spelbrink, J.N., Li, F.Y., Tiranti, V., Nikali, K., Yuan, Q.P.,
Supplementary Material Tariq, M., Wanrooij, S., Garrido, N., Comi, G., Morandi, L. et al.
(2001) Human mitochondrial DNA deletions associated with
Supplementary Material is available at HMG online. mutations in the gene encoding Twinkle, a phage T7 gene 4-
like protein localized in mitochondria. Nat. Genet., 28,
223–231.
Acknowledgements 13. Van Goethem, G., Dermaut, B., Löfgren, A., Martin, J.J. and
We thank all affected individuals and their families for their Van Broeckhoven, C. (2001) Mutation of POLG is associated
participation and for providing important samples for the pre- with progressive external ophthalmoplegia characterized by
sent research study. mtDNA deletions. Nat. Genet., 28, 211–212.
14. Pagnamenta, A.T., Taanman, J.-W., Wilson, C.J., Anderson,
Conflict of Interest statement. None declared. N.E., Marotta, R., Duncan, A.J., Bitner-Glindzicz, M., Taylor,
R.W., Laskowski, A., Thorburn, D.R. et al. (2006) Dominant in-
heritance of premature ovarian failure associated with mu-
Funding tant mitochondrial DNA polymerase gamma. Hum. Reprod.,
This study was supported by the Swedish Research Council 21, 2467–2473.
[A.W. (VR521-2012-2571), A.We. (VR521-2013-2341) and M.F. 15. Van Goethem, G., Martin, J.J. and Löfgren, A. (1997) Unusual
(VR521-2013-3621)]; Stockholm County Council [A.W. (K0176- presentation and clinical variability in Belgian pedigrees
2012) and A.We. (20140053)]; Swedish Foundation for Strategic with progressive external ophthalmoplegia and multiple de-
Research [A.W. (ICA 12-0017)]; Knut & Alice Wallenberg letions of mitochondrial DNA. Eur. J. Neurol., 4, 476–484.
Foundation [A.W. and A.We. (KAW 2013.0026) and M.F. (KAW 16. Lim, S.E., Ponamarev, M.V., Longley, M.J. and Copeland, W.C.
2011 and KAW 2014]); The Swedish Cancer Foundation [M.F. (2003) Structural determinants in human DNA polymerase
(CAN 2016/816]; European Research Council [M.F.]; The Swedish gamma account for mitochondrial toxicity from nucleoside
Brain Foundation [A.We. (FO2015-0146)]. A.W. is a Ragnar analogs. J. Mol. Biol., 329, 45–57.
Söderberg fellow in Medicine (M77/13). Funding to pay the Open 17. Graziewicz, M.A., Longley, M.J., Bienstock, R.J., Zeviani, M.
Access publication charges for this article was provided by the and Copeland, W.C. (2004) Structure-function defects of hu-
Swedish Research Council (VR521-2012-2571). man mitochondrial DNA polymerase in autosomal domi-
nant progressive external ophthalmoplegia. Nat. Struct. Mol.
Biol., 11, 770–776.
References 18. Lewis, W., Day, B.J., Kohler, J.J., Hosseini, S.H., Chan, S.S.L.,
1. Lee, Y.-S., Kennedy, W.D. and Yin, Y.W. (2009) Structural in- Green, E.C., Haase, C.P., Keebaugh, E.S., Long, R., Ludaway, T.
sight into processive human mitochondrial DNA synthesis et al. (2007) Decreased mtDNA, oxidative stress,
 Human Molecular Genetics, 2017, Vol. 26, No. 13 | 2525

cardiomyopathy, and death from transgenic cardiac tar- 32. Uusimaa, J., Gowda, V., McShane, A., Smith, C., Evans, J.,
geted human mutant polymerase gamma. Lab. Invest., 87, Shrier, A., Narasimhan, M., O’Rourke, A., Rajabally, Y.,
326–335. Hedderly, T., Cowan, F., Fratter, C. et al. (2013) Prospective
19. Graziewicz, M.A., Bienstock, R.J. and Copeland, W.C. (2007) study of POLG mutations presenting in children with intrac-
The DNA polymerase gamma Y955C disease variant associ- table epilepsy: prevalence and clinical features. Epilepsia, 54,
ated with PEO and parkinsonism mediates the incorporation 1002–1011.
and translesion synthesis opposite 7,8-dihydro-8-oxo-2’- 33. Kool, E.T. (2002) Active site tightness and substrate fit in
deoxyguanosine. Hum. Mol. Genet., 16, 2729–2739. DNA replication. Annu. Rev. Biochem., 71, 191–219.
20. Estep, P.A. and Johnson, K.A. (2011) Effect of the Y955C muta- 34. Shoichet, B.K., Baase, W.A., Kuroki, R. and Matthews, B.W.
tion on mitochondrial DNA polymerase nucleotide incorpo- (1995) A relationship between protein stability and protein
ration efficiency and fidelity. Biochemistry, 50, 6376–6386. function. Proc. Natl. Acad. Sci. USA, 92, 452–456.
21. Atanassova, N., Fusté, J.M., Wanrooij, S., Macao, B., Goffart, 35. Merski, M. and Shoichet, B.K. (2012) Engineering a model
S., Ba € ckström, S., Farge, G., Khvorostov, I., Larsson, N.-G., protein cavity to catalyze the Kemp elimination. Proc. Natl.
Spelbrink, J.N. et al. (2011) Sequence-specific stalling of DNA Acad. Sci. USA, 109, 16179–16183.
polymerase c and the effects of mutations causing progres- 36. Merski, M. and Shoichet, B.K. (2013) The impact of introduc-
sive ophthalmoplegia. Hum. Mol. Genet., 20, 1212–1223. ing a histidine into an apolar cavity site on docking and li-
22. Luoma, P.T., Luo, N., Löscher, W.N., Farr, C.L., Horva  th, R., gand recognition. J. Med. Chem., 56, 2874–2884.
Wanschitz, J., Kiechl, S., Kaguni, L.S. and Suomalainen, A. 37. Stuart, G.R., Santos, J.H., Strand, M.K., van Houten, B. and
(2005) Functional defects due to spacer-region mutations of Copeland, W.C. (2006) Mitochondrial and nuclear
human mitochondrial DNA polymerase in a family with an DNA defects in Saccharomyces cerevisiae with mutations
ataxia-myopathy syndrome. Hum. Mol. Genet., 14, 1907–1920. in DNA polymerase gamma associated with progres-
23. Stranneheim, H., Engvall, M., Naess, K., Lesko, N., Larsson, sive external ophthalmoplegia. Hum. Mol. Genet., 15,
P., Dahlberg, M., Andeer, R., Wredenberg, A., Freyer, C., 363–374.
Barbaro, M. et al. (2014) Rapid pulsed whole genome se- 38. Baruffini, E., Lodi, T., Dallabona, C., Puglisi, A., Zeviani, M.
quencing for comprehensive acute diagnostics of inborn er- and Ferrero, I. (2006) Genetic and chemical rescue of the
rors of metabolism. BMC Genomics, 15, 1090. Saccharomyces cerevisiae phenotype induced by mitochon-
24. Freyer, C., Stranneheim, H., Naess, K., Mourier, A., Felser, A., drial DNA polymerase mutations associated with progres-
Maffezzini, C., Lesko, N., Bruhn, H., Engvall, M., Wibom, R. sive external ophthalmoplegia in humans. Hum. Mol. Genet.,
et al. (2015) Rescue of primary ubiquinone deficiency due to a 15, 2846–2855.
novel COQ7 defect using 2,4-dihydroxybensoic acid. J. Med. 39. Ponamarev, M.V., Longley, M.J., Nguyen, D., Kunkel, T.A. and
Genet., 52, 779–783. Copeland, W.C. (2002) Active site mutation in DNA polymer-
25. Bratic, A., Kauppila, T.E.S., Macao, B., Grönke, S., Siibak, T., ase gamma associated with progressive external ophthal-
Stewart, J.B., Baggio, F., Dols, J., Partridge, L., Falkenberg, M. moplegia causes error-prone DNA synthesis. J. Biol. Chem.,
et al. (2015) Complementation between polymerase- and 277, 15225–15228.
exonuclease-deficient mitochondrial DNA polymerase mu- 40. Qian, Y., Kachroo, A.H., Yellman, C.M., Marcotte, E.M. and
tants in genomically engineered flies. Nat. Commun., 6, 8808. Johnson, K.A. (2014) Yeast cells expressing the human mito-
26. Korhonen, J.A., Pham, X.H., Pellegrini, M. and Falkenberg, M. chondrial DNA polymerase reveal correlations between po-
(2004) Reconstitution of a minimal mtDNA replisome in vitro. lymerase fidelity and human disease progression. J. Biol.
EMBO J., 23, 2423–2429. Chem., 289, 5970–5985.
27. Song, S., Pursell, Z.F., Copeland, W.C., Longley, M.J., Kunkel, 41. Tyynismaa, H., Mjosund, K.P., Wanrooij, S., Lappalainen, I.,
T.A. and Mathews, C.K. (2005) DNA precursor asymmetries Ylikallio, E., Jalanko, A., Spelbrink, J.N., Paetau, A. and
in mammalian tissue mitochondria and possible contribu- Suomalainen, A. (2005) Mutant mitochondrial helicase
tion to mutagenesis through reduced replication fidelity. Twinkle causes multiple mtDNA deletions and a late-onset
Proc. Natl. Acad. Sci. USA, 102, 4990–4995. mitochondrial disease in mice. Proc. Natl. Acad. Sci. USA, 102,
28. Gandhi, V.V. and Samuels, D.C. (2011) A review comparing 17687–17692.
deoxyribonucleoside triphosphate (dNTP) concentrations in 42. Taylor, R.W., Taylor, G.A., Durham, S.E. and Turnbull, D.M.
the mitochondrial and cytoplasmic compartments of nor- (2001) The determination of complete human mitochondrial
mal and transformed cells. Nucleosides Nucleotides Nucleic DNA sequences in single cells: implications for the study of
Acids, 30, 317–339. somatic mitochondrial DNA point mutations. Nucleic Acids
29. Macao, B., Uhler, J.P., Siibak, T., Zhu, X., Shi, Y., Sheng, W., Res., 29, E74–E74.
Olsson, M., Stewart, J.B., Gustafsson, C.M. and Falkenberg, M. 43. Naess, K., Barbaro, M., Bruhn, H., Wibom, R., Nennesmo, I.,
(2015) The exonuclease activity of DNA polymerase c is re- von Döbeln, U., Larsson, N.-G., Nemeth, A. and Lesko, N.
quired for ligation during mitochondrial DNA replication. (2012) Complete deletion of a POLG1 allele in a patient with
Nat. Commun., 6, 7303. Alpers syndrome. JIMD Rep., 4, 67–73.
30. Zeviani, M., Moraes, C.T., DiMauro, S., Nakase, H., Bonilla, E., 44. Larsson, N.-G., Holme, E., Kristiansson, B., Oldfors, A. and
Schon, E.A. and Rowland, L.P. (1988) Deletions of mitochondrial Tulinius, M. (1990) Progressive increase of the mutated mito-
DNA in Kearns-Sayre syndrome. Neurology, 38, 1339–1346. chondrial DNA fraction in Kearns-Sayre syndrome. Pediatr
31. Kurt, B., Jaeken, J., Van Hove, J., Lagae, L., Löfgren, A., Res., 28, 131–136.
Everman, D.B., Jayakar, P., Naini, A., Wierenga, K.J., Van 45. Huang, J., Zhou, W., Dong, W., Watson, A.M. and Hong, Y.
Goethem, G., Copland, W.C. and DiMauro, S. (2010) A novel (2009) From the Cover: Directed, efficient, and versatile mod-
POLG gene mutation in 4 children with Alpers-like hepato- ifications of the Drosophila genome by genomic engineering.
cerebral syndromes. Arch Neurol., 67, 239–244. Proc. Natl. Acad. Sci. USA, 106, 8284–8289.
 1

2
ol

ol
ct

ct
B)

ntr

ntr
bj e

bj e
A)

Co

Co
Su

Su
16.5kb

C) D)

1,2 1,6
ND1 ND1
ND5 1,4 ND5
1,0
Relative mtDNA levels

Relative mtDNA levels

1,2
0,8
1,0

0,6 0,8

0,6
0,4
0,4
0,2
0,2

0,0 0,0
Controls Subject 1 Controls Subject 2

Figure S1. (A) Electron-microsope image of skeletal muscle from subject 1, showing parac-
rystalline inclusions (scale:XX). (B) Southern blot analysis of total DNA from skeletal
muscle from subjects 1 and 2, using XX as probe. Quantification of mtDNA copy number
by qPCR in skeletal muscle samples from (C) subject 1 and (D) subject 2, using TaqMan
probes against mitochondrially encoded NADH dehydrogenase subunits 1 (ND1) and 5
(ND5). Values were normalised to the nuclear encoded 18S gene and compared to
age-matched Control samples (N=5 for subject 1 and N=2 for subject 2). Error bars are
±SD.
 A)
2L
Endogenous b tam sop2

A B C D

2L
Knock-in b attR tam w attL sop2

A B E C D

B) C)
A+B C+D+E

Y8 H/+

/+
C
73 /+
/+

Y8 H/+

/+

73

73

t
ah

t/w
t/+
Y8 3H
C

wD

Y8
73
73
ah

ah

w
t/+

t/+
7
wD

Mw wD
Y8

Y8
Mw
w

Adult flies Adult flies Adult flies

D)

1,030 1,040 1,050

CG8987

wt/+

Y873H/+

Y873C/+

Figure S2. Targeting of the TAMAS locus in Drosophila melanogaster. (A) Schematic diagram of the TAMAS
locus and genomic primers in control (endogenous) and targeted (knock-in) flies. (B) PCR to confirm target-
ing, using primers as indicated in (A). (C) Southern blot analsysis confirming correct targeting of the mutant
TAMAS constructs. (D) Electropherogram of the Y873 position in control (tamas/+) and knock-in (Y873H/+,
and Y873C/+) flies.
A) B)

1 1

0.8 0.8
survival

0.6

survival
0.6
F1
0.4 0.4 F6
WT WT
0.2 Y873C/+ 0.2 Y873C/+
Y873H/+ Y873H/+
0 0
10 20 30 40 50 60 70 80 90 100 0 10 20 30 40 50 60 70 80 90 100
days days

C) 1

0.8

0.6
survival

0.4 F15
WT
0.2 Y873C/+
Y873H/+
0
0 10 20 30 40 50 60 70 80 90 100
days

Figure S3: Life span. Survival curves of control (black), heterozygous Y873C/+ (red) and
Y873H/+ flies (green), grown on standard food. Life spans were measured after (A) first
generation (B) 6 generations and (C) 15 generations intercrossing.
A)
WT Y955H Y955C

00
0

00
0

00
0

0
0
30 30

0
Amount (fmol) 30 0 1 3 10 30 0 1 3 10 30

10
0 1 3 10 30

10
10

10
10

10
Bound DNA

Free DNA

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

B)
POLγ DNA binding Kd (nM)
WT 4,5 ± 0,6
Y955H 26,9 ± 0,1
Y955C 5,0 ± 1,1

Figure S4. (A) Representative gels showing electrophoretic mobility assays using WT and
mutant POLγ holoenzymes (in complex with polγB) to estimate the Kd (DNA) values. Each
lane contains 10 fmol of DNA substrate and the indicated amounts of protein complex in a
reaction volume of 15µl. (B) DNA binding Kd. Values for dissociation constants are shown as
an average from at least three independent binding assays. Errors are presented as stan-
dard deviations.
Table S1: Mutation frequency in skeletal muscle samples. Skeletal muscle DNA from subjects 1 and 2, as well as age-matched control
samples were amplified by PCR, followed by cloning and Sanger sequencing.

Sample Genotype Number of base Number of Mutations identified Mutation frequency Frequency / 100,000
pairs sequenced mutations

control 1 p.Y955 123843 17 m.10257C>A; m.10242dupATTACCT; m.9863C>A; m.10614C>A; 1 / 7285 bases 14

m.10202C>T; m.10623C>A; m.10280C>A; m.10047C>A; m.
10592C>A; m.10398A>G; m.10431G>T; m.10196C>A; m.
9826A>G; m.10152T>G; m.10092C>A; m.10194C>A; m.
10331C>A
subject 1 p.Y955C 119440 12 m.10195C>A; m.10254G>T;m.10024 A>G; m.10100 C>A; m.9931 1 / 9954 bases 10
G>T; m.10186C>T; m.10223C>A; m.10166T>C; m.10629C>A; m.
9806C>A; m.10013C>A; m.10332C>A
control 2 p.Y955 92597 0 0

subject 2 p.Y955H 98666 1 m.10357 T>C 1 / 98666 bases 1