Mitochondrial DNA (mtDNA) is believed to be highly vulnerable to age-associated damage and mutagenesis by reactive oxygen species (ROS). However, somatic mtDNA mutations have historically been difficult to study because of technical limitations in accurately quantifying rare mtDNA mutations. We have applied the highly sensitive Duplex Sequencing methodology, which can detect a single mutation among >107 wild type molecules, to sequence mtDNA purified from human brain tissue from both young and old individuals with unprecedented accuracy. We find that the frequency of point mutations increases ∼5-fold over the course of 80 years of life. Overall, the mutation spectra of both groups are comprised predominantly of transition mutations, consistent with misincorporation by DNA polymerase γ or deamination of cytidine and adenosine as the primary mutagenic events in mtDNA. Surprisingly, G→T mutations, considered the hallmark of oxidative damage to DNA, do not significantly increase with age. We observe a non-uniform, age-independent distribution of mutations in mtDNA, with the D-loop exhibiting a significantly higher mutation frequency than the rest of the genome. The coding regions, but not the D-loop, exhibit a pronounced asymmetric accumulation of mutations between the two strands, with G→A and T→C mutations occurring more often on the light strand than the heavy strand. The patterns and biases we observe in our data closely mirror the mutational spectrum which has been reported in studies of human populations and closely related species. Overall our results argue against oxidative damage being a major driver of aging and suggest that replication errors by DNA polymerase γ and/or spontaneous base hydrolysis are responsible for the bulk of accumulating point mutations in mtDNA.
Vyšlo v časopise: Ultra-Sensitive Sequencing Reveals an Age-Related Increase in Somatic Mitochondrial Mutations That Are Inconsistent with Oxidative Damage. PLoS Genet 9(9): e32767. doi:10.1371/journal.pgen.1003794 Kategorie: Research Article prolekare.web.journal.doi_sk: https://doi.org/10.1371/journal.pgen.1003794
1. HarmanD (1956) Aging: A theory based on free radical and radiation chemistry. J Gerontol 11 : 298–300.
2. HarmanD (1972) The biological clock: The mitochondria? J Am Geriatr Soc 20 : 145–147.
3. WallaceDC (1999) Mitochondrial diseases in man and mouse. Science 283 : 1482–1488.
4. TaylorRW, TurnbullDM (2005) Mitochondrial DNA mutations in human disease. Nat Rev Genet 6 : 389–402.
5. ShortKR, BigelowML, KahlJ, SinghR, Coenen-SchimkeJ, et al. (2005) Decline in skeletal muscle mitochondrial function with aging in humans. Proc Nat Acad Sci U S A 102 : 5618–5623.
6. GreavesLC, BarronMJ, Campbell-ShielG, KirkwoodTBL, TurnbullDM (2011) Differences in the accumulation of mitochondrial defects with age in mice and humans. Mech Ageing Devel 132 : 588–591.
7. CaoZ, WanagatJ, McKiernanSH, AikenJM (2001) Mitochondrial DNA deletion mutations are concomitant with ragged red regions of individual, aged muscle fibers: analysis by laser-capture microdissection. Nuc Acid Res 29 : 4502–4508.
8. HsiehR, HouJ, HsuH, WeiW (1994) Age-dependent respiratory function decline and DNA deletions in human muscle mitochondria. Biochem Mol Biol Int 32 : 1009–1022.
9. KraytsbergY, KudryavtsevaE, McKeeAC, GeulaC, KowallNW, et al. (2006) Mitochondrial DNA deletions are abundant and cause functional impairment in aged human substantia nigra neurons. Nat Genetics 38 : 518–520.
10. LezzaA, BoffoliD, CantatoreP, GadaletaM (1994) Correlation between mitochondrial DNA 4977-bp deletion and respiratory chain enzyme activities in aging human skeletal muscles. Biochem Bioph Res Co 205 : 772–779.
11. Corral-DebrinskiM, HortonT, LottMT, ShoffnerJM, BealMF, et al. (1992) Mitochondrial DNA deletions in human brain: Regional variability and increase with advanced age. Nat Genetics 2 : 324–329.
12. MohamedSA, HankeT, ErasmiAW, BechtelMJF, ScharfschwerdtM, et al. (2006) Mitochondrial DNA deletions and the aging heart. Exp Gerontol 41 : 508–517.
13. BenderA, KrishnanKJ, MorrisCM, TaylorGA, ReeveAK, et al. (2006) High levels of mitochondrial DNA deletions in substantia nigra neurons in aging and Parkinson disease. Nat Genetics 38 : 515–517.
14. GuG, ReyesPF, GoldenGT, WoltjerRL, HuletteC, et al. (2002) Mitochondrial DNA deletions/rearrangements in parkinson disease and related neurodegenerative disorders. J Neuropath Exp Neurol 61 : 634–639.
15. Corral-DebrinskiM, HortonT, LottMT, ShoffnerJM, McKeeAC, et al. (1994) Marked changes in mitochondrial DNA deletion levels in Alzheimer brains. Genomics 23 : 471–476.
16. KasaiH, NishimuraS (1984) Hydroxylation of deoxyguanosine at the C-8 position by ascorbic acid and other reducing agents. Nuc Acid Res 12 : 2137–2145.
17. FragaCG, ShigenagaMK, ParkJ-W, DeganP, AmesBN (1990) Oxidative damage to DNA during aging: 8-Hydroxy-2′-deoxyguanosine in rat organ DNA and urine. Proc Nat Acad Sci U S A 87 : 4533–4537.
18. MecocciP, MacGarveyU, KaufmanAE, KoontzD, ShoffnerJM, et al. (1993) Oxidative damage to mitochondrial DNA shows marked age-dependent increases in human brain. Ann Neurol 34 : 609–616.
19. AsuncionJGdl, MillanA, PlaR, BruseghiniL, EsterasA, et al. (1996) Mitochondrial glutathione oxidation correlates with age-associated oxidative damage to mitochondrial DNA. FASEB J 10 : 333–338.
20. BarjaG, HerreroA (2000) Oxidative damage to mitochondrial DNA is inversely related to maximum life span in the heart and brain of mammals. FASEB J 14 : 312–318.
21. EdgarD, ShabalinaI, CamaraY, WredenbergA, CalvarusoMA, et al. (2009) Random point mutations with major effects on protein-coding genes are the driving force behind premature aging in mtDNA mutator mice. Cell Metab 10 : 131–138.
22. VermulstM, BielasJH, KujothGC, LadigesWC, RabinovitchPS, et al. (2007) Mitochondrial point mutations do not limit the natural lifespan of mice. Nat Genetics 39 : 540–543.
23. LinMT, Cantuti-CastelvetriI, ZhengK, JacksonKE, TanYB, et al. (2012) Somatic mitochondrial DNA mutations in early Parkinson and incidental Lewy Body Disease. Ann Neurol 71 : 850–854.
24. LinMT, SimonDK, AhnCH, KimLM, BealMF (2002) High aggregate burden of somatic mtDNA point mutations in aging and Alzheimer's disease brain. Hum Mol Genet 11 : 133–145.
25. NekhaevaE, BodyakND, KraytsbergY, McGrathSB, OrsouwNJV, et al. (2002) Clonally expanded mtDNA point mutations are abundant in individual cells of human tissues. Proc Nat Acad Sci U S A 99 : 5521–5526.
26. MichikawaY, MazzucchelliF, BresolinN, ScarlatoG, AttardiG (1999) Mutations in the human mtDNA control region for replication. Science 286 : 774–779.
27. LoebLA (2011) Human cancers express mutator phenotypes: Origin, consequences and targeting. Nat Rev Cancer 11 : 450–457.
28. SalkJJ, FoxEJ, LoebLA (2010) Mutational heterogeneity in human cancers: Origin and consequences. Annu Rev Pathol-Mech 5 : 51–75.
29. VermulstM, BielasJH, LoebLA (2008) Quantification of random mutations in the mitochondrial genome. Methods 46 : 263–268.
30. MarcelinoLA, ThillyWG (1999) Mitochondrial mutagenesis in human cells and tissues. Mutat Res 434 : 177–203.
31. HarismendyO, NgPC, StrausbergRL, WangX, StockwellTB, et al. (2009) Evaluation of next generation sequencing platforms for population targeted sequencing studies. Genome Biol 10: R32.
32. SchmittMW, KennedySR, SalkJJ, FoxEJ, HiattJB, et al. (2012) Detection of ultra-rare mutations by next-generation sequencing. Proc Nat Acad Sci U S A 109 : 14508–14513.
33. MarinhoANdR, MoraesMRd, SantosS, Ribeiro-dos-Santos (2011) Human aging and somatic point mutations in mtDNA: A comparative study of generational differences (grandparents and grandchildren). Genet Mol Biol 34 : 31–34.
34. HeY, WuJ, DressmanDC, Iacobuzio-DonahueC, MarkowitzSD, et al. (2010) Heteroplasmic mitochondrial DNA mutations in normal and tumour cells. Nature 464 : 610–614.
35. DuncanBK, MillerJH (1980) Mutagenic deamination of cytosine residues in DNA. Nature 287 : 560–561.
36. SpelbrinkJN, ToivonenJM, HakkaartGAJ, KurkelaJM, CooperHM, et al. (2000) In vivo functional analysis of the human mitochondrial DNA polymerase POLG expressed in cultured human cells. J Biol Chem 275 : 24818–24828.
37. NordmannPL, MakrisJC, ReznikoffWS (1988) Inosine induced mutations. Mol Gen Genet 214 : 62–67.
38. SongS, PursellZF, CopelandWC, LongleyMJ, KunkelTA, et al. (2005) DNA precursor asymmetries in mammalian tissue mitochondria and possible contribution to mutagenesis through reduced replication fidelity. Proc Nat Acad Sci U S A 102 : 4990–4995.
39. LongleyMJ, NguyenD, KunkelTA, CopelandWC (2001) The fidelity of human DNA polymerase γ with and without exonucleolytic proofreading and the p55 accessory subunit. J Biol Chem 276 : 38555–38562.
40. ZhengW, KhrapkoK, CollerHA, ThillyWG, CopelandWC (2006) Origins of human mitochondrial point mutations as DNA polymerase γ-mediated errors. Mutat Res-Fundamental and Molecular Mechanisms of Mutagenesis 599 : 11–20.
41. MecocciP, MacGarveyU, BealMF (1994) Oxidative damage to mitochondrial DNA is increased in Alzheimer's disease. Ann Neurol 36 : 747–751.
42. ChengKC, CahillDS, KasaisH, NishimurasS, LoebLA (1992) 8-Hydroxyguanine, an abundant form of oxidative DNA damage, causes G→T and A→C substitutions. J Biol Chem 267 : 166–172.
43. BeckmanKB, AmesBN (1998) The free radical theory of aging matures. Physiol Rev 78 : 547–581.
44. KirkwoodTBL, KowaldA (2012) The free-radical theory of ageing-older, wiser and still alive. Bioessays 34 : 692–700.
45. LiB, KrishnanVG, MortME, XinF, KamatiKK, et al. (2009) Automated inference of molecular mechanisms of disease from amino acid substitutions. Bioinformatics 25 : 2744–2750.
46. GreavesLC, ElsonJL, NooteboomM, GradyJP, TaylorGA, et al. (2012) Comparison of mitochondrial mutation spectra in ageing human colonic epithelium and disease: Absence of evidence for purifying selection in somatic mitochondrial DNA point mutations. Plos Genet 8: e10003082.
47. GaltierN, EnardD, RadondyY, BazinE, BelkhirK (2006) Mutation hot spots in mammalian mitochondrial DNA. Genome Res 16 : 215–222.
48. MeyerS, WeissG, HaeselerAv (1999) Pattern of nucleotide substitution and rate heterogeneity in the hypervariable regions I and II of human mtDNA. Genetics 152 : 1103–1110.
49. WakeleyJ (1993) Substitution rate variation among sites in hypervariable region 1 of human mitochondrial DNA. J Mol Evol 37 : 613–623.
50. ReyesA, GissiC, PesoleG, SacconeC (1998) Asymmetrical directional mutation pressure in the mitochondrial genome of mammals. Mol Biol Evol 15 : 957–966.
51. BelleEMS, PiganeauG, GardnerM, Eyre-WalkerA (2005) An investigation of the variation in the transition bias among various animal mitochondrial DNA. Gene 355 : 58–66.
52. TanakaM, OzawaT (1994) Strand asymmetry in human mitochondrial DNA mutations. Genomics 22 : 327–335.
53. XiaX (2012) DNA replication and strand asymmetry in prokaryotic and mitochondrial genomes. Curr Genomics 13 : 16–27.
54. KennedySR, LoebLA, HerrAJ (2012) Somatic mutations in aging, cancer and neurodegeneration. Mech Ageing Dev 133 : 118–126.
55. CookeMS, EvansMD, DizdarogluM, LunecJ (2003) Oxidative DNA damage: mechanisms, mutation, and disease. FaSEB J 17 : 1195–1214.
56. MarnettLJ (2000) Oxyradicals and DNA damage. Carcinogenesis 21 : 361–370.
57. AmeurA, StewartJB, FreyerC, HagströmE, IngmanM, et al. (2011) Ultra-deep sequencing of mouse mitochondrial DNA: Mutational patterns and their origins. Plos Genet 7: e1002028.
58. HalsneR, EsbensenY, WangW, SchefflerK, SuganthanR, et al. (2012) Lack of the DNA glycosylases MYH and OGG1 in the cancer prone double mutant mouse does not increase mitochondrial DNA mutagenesis. DNA Repair 11 : 278–285.
59. Ruiz-PesiniE, LottMT, ProcaccioV, PooleJC, BrandonMC, et al. (2007) An enhanced MITOMAP with a global mtDNA mutational phylogeny. Nuc Acids Res 35: D823–D828.
60. KocherTD, ThomasWK, MeyerA, EdwardsSV, PååboS, et al. (1989) Dynamics of mitochondrial DNA evolution in animals: amplification and sequencing with conserved primers. Proc Nat Acad Sci U S A 86 : 6196–6200.
61. BasuAK, LoechlerEL, LeadonSA, EssigmannJM (1989) Genetic effects of thymine glycol: Site-specific mutagenesis and molecular modeling studies. Proc Nat Acad Sci U S A 86 : 7677–7681.
62. KreutzerDA, EssigmannJM (1998) Oxidized, deaminated cytosines are a source of C→T transitions in vivo. Proc Nat Acad Sci U S A 95 : 3578–3582.
63. Souz-PintoNC, CroteauDL, HudsonEK, HansfordRG, BohrVA (1999) Age-associated increase in 8-oxo-deoxyguanosine glycosylase/AP lyase activity in rat mitochondria. Nuc Acid Res 27 : 1935–1942.
64. FurdaAM, MarrangoniAM, LokshinA, HoutenBV (2012) Oxidants and not alkylating agents induce rapid mtDNA loss and mitochondrial dysfunction. DNA Repair 11 : 684–692.
65. YangYX, MuqitMMK, LatchmanDS (2006) Induction of parkin expression in the presence of oxidative stress. Eur J Neurosci 24 : 1366–1372.
66. HanesJW, ThalDM, JohnsonKA (2006) Incorporation and replication of 8-oxo-deoxyguanosine by the human mitochondrial DNA polymerase. J Biol Chem 281 : 36241–36248.
67. BrownTA, CecconiC, TkachukAN, BustamanteC, ClaytonDA (2005) Replication of mitochondrial DNA occurs by strand displacement with alternative light-strand origins, not via a strand-coupled mechanism. Genes Dev 19 : 2466–2476.
68. ClaytonDA (1982) Replication of animal mitochondrial DNA. Cell 28 : 693–705.
69. FredericoLA, KunkelTA, ShawBR (1990) A sensitive genetic assay for the detection of cytosine deamination: Determination of rate constants and the activation energy. Biochemistry 29 : 2532–2537.
70. LiH, DurbinR (2009) Fast and accurate short read alignment with Burrows–Wheeler transform. Bioinformatics 25 : 1754–1760.
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