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Cited by 173Mitochondrial Function and PathologyDNA Repair MechanismsMetabolism and Genetic Disorders

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TWINKLE is an essential mitochondrial helicase required for synthesis of nascent D-loop strands and complete mtDNA replication

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Milenkovic, D., Matic, S., Kuhl, I., Ruzzenente, B., Freyer, C., Jemt, E., Park, C. B., Falkenberg, M., Larsson, N.-G.·2013·Human Molecular Genetics
DOI 10.1093/hmg/ddt051PMC PMC3633371
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Abstract

Replication of the mammalian mitochondrial DNA (mtDNA) is dependent on the minimal replisome, consisting of the heterotrimeric mtDNA polymerase (POLG), the hexameric DNA helicase TWINKLE and the tetrameric single-stranded DNA-binding protein (mtSSB). TWINKLE has been shown to unwind DNA during the replication process and many disease-causing mutations have been mapped to its gene. Patients carrying Twinkle mutations develop multiple deletions of mtDNA, deficient respiratory chain function and neuromuscular symptoms. Despite its importance in human disease, it has been unclear whether TWINKLE is the only replicative DNA helicase in mammalian mitochondria. Furthermore, a substantial portion of mtDNA replication events is prematurely terminated at the end of mitochondrial control region (D-loop) and it is unknown whether TWINKLE also has a role in this abortive replication. Here, we present a conditional mouse knockout for Twinkle and demonstrate that TWINKLE is essential for mouse embryonic development and thus is the only replicative DNA helicase in mammalian mitochondria. Conditional knockout of Twinkle results in severe and rapid mtDNA depletion in heart and skeletal muscle. No replication intermediates or deleted mtDNA molecules are observed after Twinkle knockout, suggesting that TWINKLE once loaded is very processive. We also demonstrate that TWINKLE is essential for nascent H-strand synthesis in the D-loop, thus showing that there is no separate DNA helicase responsible for replication of this region. Our data thus suggest that the relative levels of abortive D-loop synthesis versus complete mtDNA replication are regulated and may provide a mechanism to control progression to complete mtDNA replication.

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Figures

Figure 1. Conditional knockout of Twinkle. (A) The targeting strategy for the conditional disruption of the Twinkle gene. (B) Morphology of heterozygous (Twinkle+/−) and homozygous knockout (Twinkle−/−) embryos at E8.5. Scale bar, 0.5 mm. (C) RT–PCR analysis of Twinkle transcripts from wild-type (L/L) and tissue-specific knockout mice (L/L, cre). (D) Heart-to-body weight ratio of control (L/L) and tissue-specific knockout mice (L/L, cre) at indicated time points. Error bars represent SEM; *P<0.05, **P<0.01, ***P<0.001, Student's t-test. (E) Western blot analysis of TWINKLE protein levels from wild-type (L/L) and tissue-specific knockout mice (L/L, cre) at different ages. VDAC levels were used as a loading control.
Figure 1. Conditional knockout of Twinkle. (A) The targeting strategy for the conditional disruption of the Twinkle gene. (B) Morphology of heterozygous (Twinkle+/−) and homozygous knockout (Twinkle−/−) embryos at E8.5. Scale bar, 0.5 mm. (C) RT–PCR analysis of Twinkle transcripts from wild-type (L/L) and tissue-specific knockout mice (L/L, cre). (D) Heart-to-body weight ratio of control (L/L) and tissue-specific knockout mice (L/L, cre) at indicated time points. Error bars represent SEM; *P<0.05, **P<0.01, ***P<0.001, Student's t-test. (E) Western blot analysis of TWINKLE protein levels from wild-type (L/L) and tissue-specific knockout mice (L/L, cre) at different ages. VDAC levels were used as a loading control.CC BY-NC · PubMed Central
Figure 2. MtDNA depletion in TWINKLE knockout mice. (A) Southern blot analysis of mtDNA levels from wild-type (L/L) and tissue-specific knockout mice (L/L, cre) at different ages. The nuclear 18S rRNA gene was used as a loading control. (B) Southern blot quantification: ratio of the mtDNA signal to 18S DNA loading control from four independent experiments. Error bars represent the SEM. (C) Long-extension PCR of heart (H) skeletal muscle (M) isolated total DNA from 12-week-old wild-type (L/L) and tissue-specific knockout mice (L/L, cre) using primers P0 and P1. A vector containing whole mtDNA was used as a control (V).
Figure 2. MtDNA depletion in TWINKLE knockout mice. (A) Southern blot analysis of mtDNA levels from wild-type (L/L) and tissue-specific knockout mice (L/L, cre) at different ages. The nuclear 18S rRNA gene was used as a loading control. (B) Southern blot quantification: ratio of the mtDNA signal to 18S DNA loading control from four independent experiments. Error bars represent the SEM. (C) Long-extension PCR of heart (H) skeletal muscle (M) isolated total DNA from 12-week-old wild-type (L/L) and tissue-specific knockout mice (L/L, cre) using primers P0 and P1. A vector containing whole mtDNA was used as a control (V).CC BY-NC · PubMed Central
Figure 3. Severely impaired D-loop formation in Twinkle knockout mice. (A) Southern blot analysis of the absolute D-loop levels of wild-type (L/L) and tissue-specific knockout mice (L/L, cre) at 16 weeks of age. The same amount of total DNA was loaded in both lanes and the same blot is shown at two different exposures. (B) Southern blot analysis of the absolute D-loop levels of wild-type (L/L) and tissue- specific knockout mice (L/L, cre) at 4 and 16 weeks of age. The same amount of total DNA was loaded in each lane. (C) Southern blot analysis of the 7S DNA levels of wild-type (L/L) and tissue-specific knockout mice (L/L, cre) at 16 weeks of age. To allow relative comparisons, the amount of loaded mtDNA from knockout sample (L/L, cre) was approximately four times higher than the amount from wild-type samples (L/L, cre).
Figure 3. Severely impaired D-loop formation in Twinkle knockout mice. (A) Southern blot analysis of the absolute D-loop levels of wild-type (L/L) and tissue-specific knockout mice (L/L, cre) at 16 weeks of age. The same amount of total DNA was loaded in both lanes and the same blot is shown at two different exposures. (B) Southern blot analysis of the absolute D-loop levels of wild-type (L/L) and tissue- specific knockout mice (L/L, cre) at 4 and 16 weeks of age. The same amount of total DNA was loaded in each lane. (C) Southern blot analysis of the 7S DNA levels of wild-type (L/L) and tissue-specific knockout mice (L/L, cre) at 16 weeks of age. To allow relative comparisons, the amount of loaded mtDNA from knockout sample (L/L, cre) was approximately four times higher than the amount from wild-type samples (L/L, cre).CC BY-NC · PubMed Central
Figure 4. Steady-state levels of mitochondrial transcripts in the absence of TWINKLE. (A) Northern blot analysis of mRNAs and rRNAs isolated from the heart tissue of control (L/L) and knockout (L/L, cre) mice at 4 weeks of age. The nuclear 18S rRNA was used as a loading control. (B) Northern blot analysis of mRNAs and rRNAs isolated from the heart tissue of control (L/L) and knockout (L/L, cre) mice at 16 weeks of age. The nuclear 18S rRNA was used as a loading control. (C) Quantification of steady-state levels of the mRNA and rRNA transcripts from wild-type (L/L) and knockout (L/L, cre) mice at different ages (age 4 weeks, n = 5 pairs of animals; 8 weeks, n = 3; 12 weeks, n = 4; 16 weeks, n = 3;). Error bars represent SEM; *P < 0.05, **P < 0.01, ***P < 0.001, Student's t-test. (D) Northern blot analysis of tRNAs isolated from the heart tissue of control (L/L) and knockout (L/L, cre) mice at 4 weeks of age. The nuclear 18S rRNA was used as a loading control. (E) Northern blot analysis of tRNAs isolated from the heart tissue of control (L/L) and knockout (L/L, cre) mice at 16 weeks of age. The nuclear 18S rRNA was used as a loading control. The autoradiographs shown in panels A and D, as well as those shown in panels B and E, originate from the same gel. Therefore, the 18S loading control is identical in those panels. (F) Quantification of steady-state levels of tRNAs from wild-type (L/L) and knockout (L/L, cre) mice at different ages (4 weeks, n = 5 pairs of animals; 8 weeks, n = 3; 12 weeks, n = 4; 16 weeks, n = 3). Error bars represent SEM; *P < 0.05, **P < 0.01, ***P < 0.001, Student's t-test.
Figure 4. Steady-state levels of mitochondrial transcripts in the absence of TWINKLE. (A) Northern blot analysis of mRNAs and rRNAs isolated from the heart tissue of control (L/L) and knockout (L/L, cre) mice at 4 weeks of age. The nuclear 18S rRNA was used as a loading control. (B) Northern blot analysis of mRNAs and rRNAs isolated from the heart tissue of control (L/L) and knockout (L/L, cre) mice at 16 weeks of age. The nuclear 18S rRNA was used as a loading control. (C) Quantification of steady-state levels of the mRNA and rRNA transcripts from wild-type (L/L) and knockout (L/L, cre) mice at different ages (age 4 weeks, n = 5 pairs of animals; 8 weeks, n = 3; 12 weeks, n = 4; 16 weeks, n = 3;). Error bars represent SEM; *P < 0.05, **P < 0.01, ***P < 0.001, Student's t-test. (D) Northern blot analysis of tRNAs isolated from the heart tissue of control (L/L) and knockout (L/L, cre) mice at 4 weeks of age. The nuclear 18S rRNA was used as a loading control. (E) Northern blot analysis of tRNAs isolated from the heart tissue of control (L/L) and knockout (L/L, cre) mice at 16 weeks of age. The nuclear 18S rRNA was used as a loading control. The autoradiographs shown in panels A and D, as well as those shown in panels B and E, originate from the same gel. Therefore, the 18S loading control is identical in those panels. (F) Quantification of steady-state levels of tRNAs from wild-type (L/L) and knockout (L/L, cre) mice at different ages (4 weeks, n = 5 pairs of animals; 8 weeks, n = 3; 12 weeks, n = 4; 16 weeks, n = 3). Error bars represent SEM; *P < 0.05, **P < 0.01, ***P < 0.001, Student's t-test.CC BY-NC · PubMed Central
Figure 5. Loss of TWINKLE leads to reduced respiratory chain assembly. (A) Western blot analysis of the steady-state levels of various mitochondrial proteins from isolated heart mitochondria of control (L/L) and knockout (L/L, cre) mice at different ages. (B) BN–PAGE analysis of mitochondria isolated from TWINKLE knockout (L/L, cre) hearts and controls (L/L) at 10 weeks of age. OXPHOS complexes were detected with subunit-specific antibodies or Coomassie Brilliant Blue staining. *-unspecific bands.
Figure 5. Loss of TWINKLE leads to reduced respiratory chain assembly. (A) Western blot analysis of the steady-state levels of various mitochondrial proteins from isolated heart mitochondria of control (L/L) and knockout (L/L, cre) mice at different ages. (B) BN–PAGE analysis of mitochondria isolated from TWINKLE knockout (L/L, cre) hearts and controls (L/L) at 10 weeks of age. OXPHOS complexes were detected with subunit-specific antibodies or Coomassie Brilliant Blue staining. *-unspecific bands.CC BY-NC · PubMed Central
Figure 6. TWINKLE overexpression increases mtDNA in BAC transgenic mice. (A) Western blot analysis of the steady-state levels of various mitochondrial proteins from isolated liver and heart mitochondria of wild-type (+/+) and transgenic (+/T) mice. (B) Southern blot analysis and quantification of mtDNA levels in total DNA extracts from liver and heart from wild-type (+/+) and transgenic (+/T) mice. The nuclear 18S rRNA gene was used as a loading control. Error bars represent SEM. (C) Western blot analysis of the steady-state levels of TWINKLE protein from isolated heart, liver and kidney mitochondria from TFAM knockout mice (L/L, cre) and their controls (L/L) and from Tfam PAC transgenic animals (+/T) and their controls (+/+). VDAC levels were used as a loading control.
Figure 6. TWINKLE overexpression increases mtDNA in BAC transgenic mice. (A) Western blot analysis of the steady-state levels of various mitochondrial proteins from isolated liver and heart mitochondria of wild-type (+/+) and transgenic (+/T) mice. (B) Southern blot analysis and quantification of mtDNA levels in total DNA extracts from liver and heart from wild-type (+/+) and transgenic (+/T) mice. The nuclear 18S rRNA gene was used as a loading control. Error bars represent SEM. (C) Western blot analysis of the steady-state levels of TWINKLE protein from isolated heart, liver and kidney mitochondria from TFAM knockout mice (L/L, cre) and their controls (L/L) and from Tfam PAC transgenic animals (+/T) and their controls (+/+). VDAC levels were used as a loading control.CC BY-NC · PubMed Central

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Authors
Milenkovic, D., Matic, S., Kuhl, I., Ruzzenente, B., Freyer, C., Jemt, E., Park, C. B., Falkenberg, M., & Larsson, N.-G.
Affiliations
  • Max Planck Institute for Biology of Ageing (DE)
  • Karolinska Institutet (SE)
  • University of Gothenburg (SE)
Corresponding author
Larsson, N.-G.
ORCID
Venue
Human Molecular Genetics
Publisher
Oxford University Press (OUP)
Published
2013-02-07
Volume
22
Issue
10
Pages
1983-1993
ISSN
  • 0964-6906 (ISSN-L)
  • 1460-2083
DOI
10.1093/hmg/ddt051
PMC
PMC3633371
OpenAlex
W2167764669
Cited by
173
References
52
Citations by year
2026: 11 · 2025: 18 · 2024: 11 · 2023: 1 · 2022: 19 · 2021: 10 · 2020: 13 · 2019: 17 · 2018: 15 · 2017: 12 · 2016: 19 · 2015: 11 · 2014: 9 · 2013: 7
FWCI
6.86
Type
journal article
Language
en
Open access
Licence
cc-by-nc
Locations
  • Human Molecular Genetics (publishedVersion, cc-by-nc)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by-nc)
  • PubMed Central (publishedVersion, cc-by-nc)
  • PubMed (publishedVersion)
  • AJOU Open Repository (Ajou University) (submittedVersion, cc-by-nc)
  • MPG.PuRe (Max Planck Society) (submittedVersion)
Funders
  • Swedish Cancer Foundation
  • Vetenskapsrådet
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Mitochondrial Function and Pathology
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › DNA Repair Mechanisms
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Clinical Biochemistry › Metabolism and Genetic Disorders
Keywords
Twinkle, mitochondrial DNA replication, DNA helicase, D-loop, mtDNA depletion, conditional knockout, mouse embryo development, DNA polymerase gamma, mitochondrial DNA control region, mitochondrial disease
MeSH terms
Animals, DNA Replication, DNA Helicases, DNA, Mitochondrial, Humans, Mutation, Neuromuscular Diseases, Mice, Knockout, Mitochondrial Proteins, Genetic Diseases, Inborn, Mice

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