title: Quantitative PCR for detection of DNA damage in mitochondrial DNA of the fission yeast Schizosaccharomyces pombe.
abstract: Quantitative polymerase chain reaction (QPCR) has been employed to detect DNA damage and repair in mitochondrial DNA (mtDNA) of human and several model organisms. The assay also permits the quantitation of relative mtDNA copy number in cells. Here, we developed the QPCR assay primers and reaction conditions for the fission yeast Schizosaccharomyces pombe, an important model of eukaryote biology, not previously described. Under these conditions, long targets (approximately 10kb) in mtDNA were quantitatively amplified using 0.1ng of crude DNA templates without isolation of mitochondria and mtDNA. Quantitative detection of oxidative DNA damage in mtDNA was illustrated by using a DNA template irradiated with UVA in the presence of riboflavin. The damage to mtDNA in S. pombe cells treated with hydrogen peroxide and paraquat was also quantitatively measured. Finally, we found that mtDNA copy number in S. pombe cells increased after transition into a stationary phase and that the damage to mtDNA due to endogenous cellular processes accumulated during chronological aging.
authors.affiliation: Department of Biochemistry, Faculty of Science, Okayama University of Science, 1-1 Ridai-cho, Kita-ku, Okayama 700-0005, Japan.
authors.forename: Takanori
authors.initials: T
authors.lastname: Senoo
authors.affiliation: Department of Biochemistry, Faculty of Science, Okayama University of Science, 1-1 Ridai-cho, Kita-ku, Okayama 700-0005, Japan.
authors.forename: Mayumi
authors.initials: M
authors.lastname: Yamanaka
authors.affiliation: Department of Biochemistry, Faculty of Science, Okayama University of Science, 1-1 Ridai-cho, Kita-ku, Okayama 700-0005, Japan.
authors.forename: Atori
authors.initials: A
authors.lastname: Nakamura
authors.affiliation: Department of Biochemistry, Faculty of Science, Okayama University of Science, 1-1 Ridai-cho, Kita-ku, Okayama 700-0005, Japan.
authors.forename: Tomoki
authors.initials: T
authors.lastname: Terashita
authors.affiliation: Department of Biochemistry, Faculty of Science, Okayama University of Science, 1-1 Ridai-cho, Kita-ku, Okayama 700-0005, Japan.
authors.forename: Shinji
authors.initials: S
authors.lastname: Kawano
authors.affiliation: Department of Biochemistry, Faculty of Science, Okayama University of Science, 1-1 Ridai-cho, Kita-ku, Okayama 700-0005, Japan. Electronic address: ikeda@dbc.ous.ac.jp.
authors.forename: Shogo
authors.initials: S
authors.lastname: Ikeda
mesh_terms: D004249:DNA Damage; D017931:DNA Primers; D004271:DNA, Fungal / Q000235:genetics*; D004272:DNA, Mitochondrial / Q000235:genetics*; D018628:Gene Dosage; D006801:Humans; D006861:Hydrogen Peroxide; D008928:Mitochondria / Q000473:pathology; D016133:Polymerase Chain Reaction / Q000379:methods*; D012568:Schizosaccharomyces / Q000235:genetics*
chemical_list: D017931:DNA Primers; D004271:DNA, Fungal; D004272:DNA, Mitochondrial; D006861:Hydrogen Peroxide
keywords: DNA damage; Fission yeast; Mitochondrial DNA; Quantitative PCR; Schizosaccharomyces pombe