Abstract
Oxidative base lesions, such as 8-oxoguanine, accumulate in nuclear and mitochondrial DNAs under oxidative stress, resulting in cell death. However, it is not known whether only oxidative lesion accumulated in mitochondrial DNA is involved in such cell death. By introducing human cDNA encoding a nuclear form of 8-oxoG DNA glycosylase (hOGG1-1a) into immortalized mouse embryo fibroblasts lacking Ogg1 gene, we established a cell line which selectively accumulates 8-oxoguanine in mitochondrial DNA under oxidative stress. Selective accumulation of 8-oxoguanine in mitochondrial DNA in this cell line causes degradation of mitochondrial DNA followed by ATP depletion, mitochondrial membrane permeability transition, and Ca2+ efflux, which in turn activates calpains to execute cell death. Knockdown of MUTYH which excises adenine opposite 8-oxoG in DNA prevents degradation of mitochondrial DNA and activation of calpain, thus suppressing the cell death induced by menadione.
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References
Kasai, H. and Nishimura, S. (1984) Hydroxylation of deoxyguanosine at the C-8 position by ascorbic acid and other reducing agents. Nucleic Acids Res 12, 2137–2145.
Maki, H. (2002) Origins of spontaneous mutations: specificity and directionality of base-substitution, frameshift, and sequence-substitution mutageneses. Annu. Rev. Genet. 36, 279–303.
Shimura-Miura, H., Hattori, N., Kang, D., Miyako, K., Nakabeppu, Y. and Mizuno, Y. (1999) Increased 8-oxo-dGTPase in the mitochondria of substantia nigral neurons in Parkinson's disease. Ann. Neurol 46, 920–924.
Nunomura, A., Perry, G., Aliev, G., Hirai, K., Takeda, A., Balraj, E. K., et al. (2001) Oxidative damage is the earliest event in Alzheimer disease. J. Neuropathol. Exp. Neurol 60, 759–767.
Nakabeppu, Y., Sakumi, K., Sakamoto, K., Tsuchimoto, D., Tsuzuki, T. and Nakatsu, Y. (2006) Mutagenesis and carcinogenesis caused by the oxidation of nucleic acids. Biol. Chem 387, 373–379.
Boiteux, S. and Radicella, J. P. (2000) The human OGG1 gene: structure, functions, and its implication in the process of carcinogenesis. Arch. Biochem. Biophys 377, 1–8.
Nishioka, K., Ohtsubo, T., Oda, H., Fujiwara, T., Kang, D., Sugimachi, K., et al. (1999) Expression and differential intracellular localization of two major forms of human 8-oxoguanine DNA glycosylase encoded by alternatively spliced OGG1 mRNAs. Mol. Biol. Cell 10, 1637–1652.
Oka, S., Ohno, M., Tsuchimoto, D., Sakumi, K., Furuichi, M. and Nakabeppu, Y. (2008) Two distinct pathways of cell death triggered by oxidative damage to nuclear and mitochondrial DNAs. EMBO J 27, 421–432.
Yoshimura, D., Sakumi, K., Ohno, M., Sakai, Y., Furuichi, M., Iwai, S., et al. (2003) An oxidized purine nucleoside triphosphatase, MTH1, suppresses cell death caused by oxidative stress. J. Biol. Chem 278, 37965–37973.
Miyako, K., Takamatsu, C., Umeda, S., Tajiri, T., Furuichi, M., Nakabeppu, Y., et al. (2000) Accumulation of adenine DNA glycosylase-sensitive sites in human mitochondrial DNA. J. Biol. Chem 275, 12326–12330.
Ogden, R. C. and Adams, D. A. (1987) Electrophoresis in agarose and acrylamide gels. Methods Enzymol 152, 61–87.
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Oka, S., Ohno, M., Nakabeppu, Y. (2009). Construction and Characterization of a Cell Line Deficient in Repair of Mitochondrial, but Not Nuclear, Oxidative DNA Damage. In: Stuart, J.A. (eds) Mitochondrial DNA. Methods in Molecular Biology™, vol 554. Humana Press. https://doi.org/10.1007/978-1-59745-521-3_16
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DOI: https://doi.org/10.1007/978-1-59745-521-3_16
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