Figure 1.

mtDNA copy number in isolated mitochondria harboring HS [ori5] [rho^–] mtDNA, which were treated with various concentrations of hydrogen peroxide. (A) A physical map of the locations of the ori5-region and the probes used for detecting HS [ori5] [rho^–] mtDNA. Probe A, full length HS [ori5] [rho^–] mtDNA (1.1 kbp); probe B, a 282-bp region of ori5 and probe C, a 280-bp sequence outside of the ori5 region. The ori5 sequence (282 bp) is framed with an open box. (B) HS [ori5] [rho^–] mtDNA-containing mitochondria derived from wild-type, ntg1-null, mhr1-null, ogg1-null and cce1-null cells were suspended in incubation buffer and incubated with the indicated concentrations of hydrogen peroxide for 1 h at 26°C. HS [ori5] [rho^–] mtDNA and linearized pUC119 plasmid DNA were separated on a 1.0% agarose gel and detected by Southern blot analysis using the sequences described in (A) and linearized pUC119 as probes. Marker, 1.0-kb plus ladder. Asterisk denotes mtDNA molecules containing single-stranded regions resistant to digestion with BglII, likely with a Holliday junction structure, since they mainly appeared in HS [ori5] [rho^–] mtDNA extracted form Holliday junction resolvase-lacking cce1-null cells. (C) Quantitative analysis of the mtDNA bands in (B). Upper panel: wild type (filled diamonds); upper middle panel: ntg1-null mutant (open triangles); lower middle panel: mhr1-null mutant (open diamonds); bottom panel: ogg1-null mutant (filled squares) and lower bottom panel: cce1-null mutant (filled circles). In each panel, the colored lines representing the relative mtDNA copy number detected using Southern analysis with various probes. Black lines, probe A; red lines, probe B and blue lines, probe C. Insert in (C), a figure showing the increase of the HS [ori5] [rho^–] mtDNA copy number in isolated mitochondria treated with hydrogen peroxide concentrations ranging from 0 to 100 μM. (D) Eluted proteins from inside isolated mitochondria, accompanied by treatment with hydrogen peroxide.