Abstract
Mitochondrial DNA (mtDNA) mutations are found in several human pathologies and are associated with aging. Deletion mutations in mtDNA result in the loss of essential genes for mitochondrial function. Over 250 deletion mutations have been reported and the common deletion is the most frequent mtDNA deletion linked to disease. This deletion removes 4977 base pairs of mtDNA. It has previously been shown that exposure to UVA radiation can promote the formation of the common deletion. Furthermore, aberrations in mtDNA replication and repair are associated with formation of the common deletion. However, molecular mechanisms describing the formation of this deletion are poorly characterized. This chapter describes a method to irradiate human skin fibroblasts with physiological doses of UVA and the subsequent detection of the common deletion by quantitative PCR analysis.
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References
Dimauro S, Davidzon G (2005) Mitochondrial DNA and disease. Ann Med 37(3):222–232. https://doi.org/10.1080/07853890510007368
Russell O, Turnbull D (2014) Mitochondrial DNA disease-molecular insights and potential routes to a cure. Exp Cell Res 325(1):38–43. https://doi.org/10.1016/j.yexcr.2014.03.012
Taylor RW, Turnbull DM (2005) Mitochondrial DNA mutations in human disease. Nat Rev Genet 6(5):389–402. https://doi.org/10.1038/nrg1606
Yuan Y, Ju YS, Kim Y, Li J, Wang Y, Yoon CJ, Yang Y, Martincorena I, Creighton CJ, Weinstein JN, Xu Y, Han L, Kim HL, Nakagawa H, Park K, Campbell PJ, Liang H, Consortium P (2020) Author Correction: Comprehensive molecular characterization of mitochondrial genomes in human cancers. Nat Genet. https://doi.org/10.1038/s41588-020-0629-y
Lott MT, Leipzig JN, Derbeneva O, Xie HM, Chalkia D, Sarmady M, Procaccio V, Wallace DC (2013) mtDNA variation and analysis using Mitomap and Mitomaster. Curr Protoc Bioinformatics 44:1 23 21-26. https://doi.org/10.1002/0471250953.bi0123s44
Lu J, Sharma LK, Bai Y (2009) Implications of mitochondrial DNA mutations and mitochondrial dysfunction in tumorigenesis. Cell Res 19(7):802–815. https://doi.org/10.1038/cr.2009.69
Fontana GA, Gahlon HL (2020) Mechanisms of replication and repair in mitochondrial DNA deletion formation. Nucleic Acids Res 48(20):11244–11258. https://doi.org/10.1093/nar/gkaa804
Powers JM, Murphy G, Ralph N, O’Gorman SM, Murphy JE (2016) Mitochondrial DNA deletion percentage in sun exposed and non sun exposed skin. J Photochem Photobiol B 165:277–282. https://doi.org/10.1016/j.jphotobiol.2016.10.030
Torregrosa-Munumer R, Goffart S, Haikonen JA, Pohjoismaki JL (2015) Low doses of ultraviolet radiation and oxidative damage induce dramatic accumulation of mitochondrial DNA replication intermediates, fork regression, and replication initiation shift. Mol Biol Cell 26(23):4197–4208. https://doi.org/10.1091/mbc.E15-06-0390
Berneburg M, Grether-Beck S, Kurten V, Ruzicka T, Briviba K, Sies H, Krutmann J (1999) Singlet oxygen mediates the UVA-induced generation of the photoaging-associated mitochondrial common deletion. J Biol Chem 274(22):15345–15349. https://doi.org/10.1074/jbc.274.22.15345
Quispe-Tintaya W, White RR, Popov VN, Vijg J, Maslov AY (2013) Fast mitochondrial DNA isolation from mammalian cells for next-generation sequencing. BioTechniques 55(3):133–136. https://doi.org/10.2144/000114077
Phillips AF, Millet AR, Tigano M, Dubois SM, Crimmins H, Babin L, Charpentier M, Piganeau M, Brunet E, Sfeir A (2017) Single-molecule analysis of mtDNA replication uncovers the basis of the common deletion. Mol Cell 65(3):527–538 e526. https://doi.org/10.1016/j.molcel.2016.12.014
Acknowledgements
This work was supported by Federation of Migros Cooperatives; ETH Zurich Federation in association with the World Food System Center at ETH Zurich.
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Fontana, G.A., Gahlon, H.L. (2023). Detection of UV-Induced Deletions in Mitochondrial DNA. In: Nicholls, T.J., Uhler, J.P., Falkenberg, M. (eds) Mitochondrial DNA. Methods in Molecular Biology, vol 2615. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2922-2_20
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DOI: https://doi.org/10.1007/978-1-0716-2922-2_20
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