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Cited by 173Mitochondrial Function and PathologyGenetics, Aging, and Longevity in Model OrganismsDNA Repair Mechanisms

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Oxidative Stress Is Not a Major Contributor to Somatic Mitochondrial DNA Mutations

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Itsara, Leslie S., Kennedy, Scott R., Fox, Edward J., Yu, Selina, Hewitt, Joshua J., Sanchez-Contreras, Monica, Cardozo-Pelaez, Fernando, Pallanck, Leo J.·2014·PLoS Genetics
DOI 10.1371/journal.pgen.1003974PMC PMC3916223
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Abstract

The accumulation of somatic mitochondrial DNA (mtDNA) mutations is implicated in aging and common diseases of the elderly, including cancer and neurodegenerative disease. However, the mechanisms that influence the frequency of somatic mtDNA mutations are poorly understood. To develop a simple invertebrate model system to address this matter, we used the Random Mutation Capture (RMC) assay to characterize the age-dependent frequency and distribution of mtDNA mutations in the fruit fly Drosophila melanogaster. Because oxidative stress is a major suspect in the age-dependent accumulation of somatic mtDNA mutations, we also used the RMC assay to explore the influence of oxidative stress on the somatic mtDNA mutation frequency. We found that many of the features associated with mtDNA mutations in vertebrates are conserved in Drosophila, including a comparable somatic mtDNA mutation frequency (∼10(-5)), an increased frequency of mtDNA mutations with age, and a prevalence of transition mutations. Only a small fraction of the mtDNA mutations detected in young or old animals were G∶C to T∶A transversions, a signature of oxidative damage, and loss-of-function mutations in the mitochondrial superoxide dismutase, Sod2, had no detectable influence on the somatic mtDNA mutation frequency. Moreover, a loss-of-function mutation in Ogg1, which encodes a DNA repair enzyme that removes oxidatively damaged deoxyguanosine residues (8-hydroxy-2'-deoxyguanosine), did not significantly influence the somatic mtDNA mutation frequency of Sod2 mutants. Together, these findings indicate that oxidative stress is not a major cause of somatic mtDNA mutations. Our data instead suggests that somatic mtDNA mutations arise primarily from errors that occur during mtDNA replication. Further studies using Drosophila should aid in the identification of factors that influence the frequency of somatic mtDNA mutations.

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Figure 1 The locations of TaqI sites in Drosophila mtDNA. Schematic depiction of the Drosophila melanogaster mitochondrial genome. Numbers (1-33) indicate the different TaqI sites, color coded according to their utility for RMC analysis as indicated. Boxed numbers indicate the TaqI sites used for RMC analysis in our work. tRNA genes are represented by horizontal lines adjacent to other mitochondrial genes.
Figure 1 The locations of TaqI sites in Drosophila mtDNA. Schematic depiction of the Drosophila melanogaster mitochondrial genome. Numbers (1-33) indicate the different TaqI sites, color coded according to their utility for RMC analysis as indicated. Boxed numbers indicate the TaqI sites used for RMC analysis in our work. tRNA genes are represented by horizontal lines adjacent to other mitochondrial genes.CC BY · PubMed Central
Figure 2 Mitochondrial DNA mutation frequency increases with age and is greater in thoraces than in heads. (A) The mtDNA mutation frequency is greater in young thoraces than in young heads (**p<0.01). (B) The mtDNA mutation frequency is greater in old heads than in young heads (***p<0.001) (C) and is greater in old thoraces than in young thoraces (*p<0.05). (D) The mtDNA mutation frequency is greater in old thoraces than in old heads (**p<0.001). Horizontal bars represent the median mutation frequency, and error bars indicate the interquartile range. Significance was tested using Mann-Whitney unpaired U tests.
Figure 2 Mitochondrial DNA mutation frequency increases with age and is greater in thoraces than in heads. (A) The mtDNA mutation frequency is greater in young thoraces than in young heads (**p<0.01). (B) The mtDNA mutation frequency is greater in old heads than in young heads (***p<0.001) (C) and is greater in old thoraces than in young thoraces (*p<0.05). (D) The mtDNA mutation frequency is greater in old thoraces than in old heads (**p<0.001). Horizontal bars represent the median mutation frequency, and error bars indicate the interquartile range. Significance was tested using Mann-Whitney unpaired U tests.CC BY · PubMed Central
Figure 3 GC∶TA transversions account for only a small percentage of mtDNA mutations. The percentage and total number (in parentheses) of each type of mutation identified at TaqI sites in young and old animals. Results depict data obtained separately from heads and thoraces and also combined. The wild-type TaqI sequence (TCGA) is shown for reference. * = Two mutations identified within a single cloned TaqI site.
Figure 3 GC∶TA transversions account for only a small percentage of mtDNA mutations. The percentage and total number (in parentheses) of each type of mutation identified at TaqI sites in young and old animals. Results depict data obtained separately from heads and thoraces and also combined. The wild-type TaqI sequence (TCGA) is shown for reference. * = Two mutations identified within a single cloned TaqI site.CC BY · PubMed Central
Figure 4 Oxidative stress response pathway mutants do not have an increased mtDNA mutation frequency. (A) Lifespan of w1118 and Sod2 mutant flies. The median lifespan (indicated by dashed lines) was 60 days for w1118 flies and 41 days for Sod2 mutants. (B) The mutation frequency using pooled data from all three TaqI sites in old Sod2 mutants (39- to 41-day-old), young Ogg1ins/Ogg1ins; Sod2 double mutants (1- to 3- day-old), old Ogg1ins/Ogg1ins; Sod2 double mutants (26- to 28- day-old), and age-matched w1118 controls. The 26- to 28- day-old control estimate was obtained from linear regression of the age-dependent w1118 mutation frequency. Horizontal bars represent the median mutation frequency, and error bars indicate the interquartile range. There was no significant difference in mutation frequency between mutants and age-matched controls (Mann-Whitney unpaired U tests).
Figure 4 Oxidative stress response pathway mutants do not have an increased mtDNA mutation frequency. (A) Lifespan of w1118 and Sod2 mutant flies. The median lifespan (indicated by dashed lines) was 60 days for w1118 flies and 41 days for Sod2 mutants. (B) The mutation frequency using pooled data from all three TaqI sites in old Sod2 mutants (39- to 41-day-old), young Ogg1ins/Ogg1ins; Sod2 double mutants (1- to 3- day-old), old Ogg1ins/Ogg1ins; Sod2 double mutants (26- to 28- day-old), and age-matched w1118 controls. The 26- to 28- day-old control estimate was obtained from linear regression of the age-dependent w1118 mutation frequency. Horizontal bars represent the median mutation frequency, and error bars indicate the interquartile range. There was no significant difference in mutation frequency between mutants and age-matched controls (Mann-Whitney unpaired U tests).CC BY · PubMed Central
Figure 5 An Ogg1 mutation diminishes Ogg1 activity and interacts genetically with Sod2. (A) Amino acid sequence alignment of human Ogg1 with Drosophila Ogg1 and RpS3. Dark shading denotes amino acid identities; light shading denotes amino acid similarities. The putative N-terminal mitochondrial targeting sequence of Drosophila Ogg1 is underlined. (B) The Ogg1 gene comprises four exons, designated by rectangles, and the coding sequence is indicated with dark gray shading. The Ogg1f08013 piggyBac insertion (designated by a triangle) is located 78 base pairs upstream of the Ogg1 translation start site. (C) 8-oxo-dG repair activity was assayed by monitoring cleavage of a 32P-labeled synthetic probe containing 8-oxo-dG, using protein extracts from flies of the indicated genotypes (Df = the Df(1)BSC627 deletion that removes Ogg1; ins = the Ogg1f08013 piggyBac insertion). Results of the assay are shown following 0, 30, and 60 minutes of incubation. Neg = No protein; Pos = Mouse brain protein extract. (D) Lifespan of w1118, Ogg1ins/Ogg1ins, Sod2, and Ogg1ins/Ogg1ins; Sod2 double mutant flies. The median lifespan (indicated by dashed lines) was 60 days for w1118, 49 days for Ogg1ins mutants, 41 days for Sod2 mutants, and 31 days for Ogg1ins; Sod2 double mutants. All lifespan curves are statistically different from one another using log rank tests (p<0.001). (E) w1118, Ogg1ins/Ogg1ins, Sod2, and Ogg1ins/Ogg1ins; Sod2 double mutant flies were subjected to a mechanical stress sensitivity test at 31 days. Values represent mean time (seconds) of recovery from mechanical stress, and error bars represent standard error of the mean. Significance was determined using one-way ANOVA followed by Tukey's post hoc tests (**p<0.01; ***p<0.001).
Figure 5 An Ogg1 mutation diminishes Ogg1 activity and interacts genetically with Sod2. (A) Amino acid sequence alignment of human Ogg1 with Drosophila Ogg1 and RpS3. Dark shading denotes amino acid identities; light shading denotes amino acid similarities. The putative N-terminal mitochondrial targeting sequence of Drosophila Ogg1 is underlined. (B) The Ogg1 gene comprises four exons, designated by rectangles, and the coding sequence is indicated with dark gray shading. The Ogg1f08013 piggyBac insertion (designated by a triangle) is located 78 base pairs upstream of the Ogg1 translation start site. (C) 8-oxo-dG repair activity was assayed by monitoring cleavage of a 32P-labeled synthetic probe containing 8-oxo-dG, using protein extracts from flies of the indicated genotypes (Df = the Df(1)BSC627 deletion that removes Ogg1; ins = the Ogg1f08013 piggyBac insertion). Results of the assay are shown following 0, 30, and 60 minutes of incubation. Neg = No protein; Pos = Mouse brain protein extract. (D) Lifespan of w1118, Ogg1ins/Ogg1ins, Sod2, and Ogg1ins/Ogg1ins; Sod2 double mutant flies. The median lifespan (indicated by dashed lines) was 60 days for w1118, 49 days for Ogg1ins mutants, 41 days for Sod2 mutants, and 31 days for Ogg1ins; Sod2 double mutants. All lifespan curves are statistically different from one another using log rank tests (p<0.001). (E) w1118, Ogg1ins/Ogg1ins, Sod2, and Ogg1ins/Ogg1ins; Sod2 double mutant flies were subjected to a mechanical stress sensitivity test at 31 days. Values represent mean time (seconds) of recovery from mechanical stress, and error bars represent standard error of the mean. Significance was determined using one-way ANOVA followed by Tukey's post hoc tests (**p<0.01; ***p<0.001).CC BY · PubMed Central

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Metadata

Authors
Itsara, L. S., Kennedy, S. R., Fox, E. J., Yu, S., Hewitt, J. J., Sanchez-Contreras, M., Cardozo-Pelaez, F., & Pallanck, L. J.
Affiliations
University of Washington (US)
Corresponding author
Pallanck, Leo J.
ORCID
Venue
PLoS Genetics
Publisher
Public Library of Science (PLoS)
Published
2014-02-06
Volume
10
Issue
2
Pages
e1003974
ISSN
  • 1553-7390 (ISSN-L)
  • 1553-7404
DOI
10.1371/journal.pgen.1003974
PMC
PMC3916223
OpenAlex
W2012998724
Cited by
173
References
67
Citations by year
2026: 8 · 2025: 12 · 2024: 6 · 2023: 7 · 2022: 20 · 2021: 11 · 2020: 18 · 2019: 16 · 2018: 15 · 2017: 18 · 2016: 15 · 2015: 19 · 2014: 7
FWCI
8.07
Type
journal article
Language
en
Open access
Licence
Locations
  • PLoS Genetics (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
  • Figshare (submittedVersion, cc-by)
Funders
  • Muscular Dystrophy Association
  • National Institute on Aging (T32AG000057, P01AG001751, AG013280, P30AG013280)
  • National Cancer Institute
  • National Institute of General Medical Sciences (R01GM104990, R01GM086394)
  • National Institute of Neurological Disorders and Stroke (F31NS071857)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Mitochondrial Function and Pathology
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Aging › Genetics, Aging, and Longevity in Model Organisms
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › DNA Repair Mechanisms
Keywords
somatic mitochondrial DNA mutations, oxidative stress, aging, Drosophila melanogaster, transition mutations, transversions, OGG1, DNA repair, 8-hydroxy-2'-deoxyguanosine
MeSH terms
Superoxide Dismutase 2, Aging, Animals, DNA Repair, DNA, Mitochondrial, Drosophila melanogaster, Humans, Mitochondria, Mutation, Superoxide Dismutase, Reactive Oxygen Species, Oxidative Stress, Models, Animal, Drosophila Proteins, DNA Glycosylases, Mutation Rate

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