Changes in mtDNA copy number in isolated mitochondria treated with 10 μM hydrogen peroxide. (A) Treatment with 10 μM hydrogen peroxide increased mtDNA copy number in mitochondria isolated from wild-type cells. Isolated mitochondria were incubated with the indicated concentrations of hydrogen peroxide. mtDNA was digested with BglII and separated by electrophoresis on a 1.0% agarose gel. Marker, 1.0-kbp plus ladder. (B) Quantitative analysis of the relative mtDNA copy number in mitochondria isolated from wild-type cells. Vitamin C (500 μM), 25 μM vitamin E or 500 μM NAC were added to the incubation buffer from the start time point. After 1 h of incubation, mitochondria not treated with hydrogen peroxide were transferred into fresh incubation buffer containing 500 μM vitamin C. The average values from three independent experiments are plotted in (B). (C) Newly synthesized mtDNA (left panel) and total mtDNA (right panel) in isolated mitochondria. Mitochondria isolated from wild-type cells were incubated with DIG-dUTP, which was detected using anti-DIG AP conjugates. Total mtDNA was detected by Southern dot-blotting analysis using ³²P-labeled 1.1-kbp HS [ori5] [rho^–] mtDNA as the probe. (D) A low concentration (10 μM) of hydrogen peroxide did not induce an increase in mtDNA copy number in mitochondria isolated from either ntg1-null or mhr1-null cells. There was no change in mtDNA copy number when mitochondria isolated from either ntg1-null or mhr1-null cells were incubated with vitamin C. (E) Quantitative analysis of mtDNA copy number in mitochondria isolated from either ntg1-null or mhr1-null mutant cells. The average values from three independent experiments are plotted.

Oxidative modifications of mtDNA, which are represented by in vitro Ntg1-dependent generation of DSBs at ori5. HS [ori5] [rho^–] mtDNA was extracted from mitochondria isolated from wild-type (A) or mhr1-null (B) cells, with or without treatment with 10 μM hydrogen peroxide at 26°C for 0.5 h, and digested with BglII. In vitro cleavage by Ntg1 was carried out as described (10). Approximately 15 µg of BglII-digested DNA was incubated without (lanes 1, 3 and 5) or with 1.6 nM purified Ntg1 (lanes 2, 4 and 6) at 37°C for 0.5 h. Lanes 1 and 2, without incubation. After the removal of proteins with proteinase K, the digests were analyzed using electrophoresis on a 2.0% agarose gel. The signals representing 0.8-kbp DNA fragments derived from DSBs at ori5 and 1.1-kbp fragments corresponding to unit-size HS [ori5] [rho^–] mtDNA were detected as described in Figure 3B. The average values of the relative DSB levels at ori5 of HS [ori5] [rho^–] mtDNA from two independent experiments are shown in the top panels, which correspond to the results from the Southern blot analysis shown in the bottom panel. The ratios of the signals for the 0.8-kbp fragments were normalized against those for the 1.1-kbp fragments.

Effects of GOX expression on mtDNA copy number. (A) Western blot analysis for GOX expression. Cells harboring pVT100U or pVT100UGOX were grown at 30°C in an SD medium supplemented with required amino acid from 1.0 × 10⁵ cells/ml to 2.0 × 10⁷ cells/ml. About 10 μg of total proteins from each strain were analyzed on 10% SDS polyacrylamide gel electrophoresis. GOX and porin were detected by Western blot analysis using monoclonal antibodies. (B) Determination of hydrogen peroxide generation due to GOX expression. Cells harboring pVT100U or pVT100UGOX were grown under the same conditions as described above. The elevated levels of hydrogen peroxide caused by glucose oxidase activity were determined. Averaged values of relative hydrogen peroxide levels, which were obtained from three independent experiments, are plotted. Hydrogen peroxide level in cells harboring an empty vector was set as 1. (C) Southern blot analysis for relative mtDNA copy number. Cells harboring pVT100U or pVT100UGOX were grown under the same conditions as described in A. About 20 μg of total cellular DNA isolated as described (21) was electrophoresed on a 1.0% agarose gel. mtDNA was detected using ³²P-labeled [rho⁺] mtDNA as probes (upper panel). After the ³²P-labeled [rho⁺] mtDNA probe was washed out, the same membrane was probed using a ³²P-labeled DNA fragment containing the nuclear NUC1 gene (bottom panel). (D) Quantitative analysis of the relative amounts of mtDNA. The average values of the ratios of the signals from mtDNA versus those from the NUC1 open reading frame, obtained from three independent experiments, are plotted.

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.

DSB levels at ori5 in isolated mitochondria treated with or without 10 μM hydrogen peroxide. Isolated mitochondria were treated at 26°C with or without 10 μM hydrogen peroxide for the indicated period of time. In parallel experiments, isolated mitochondria were incubated at 26°C with 500 μM vitamin C, 25 μM vitamin E or 500 μM NAC from the start time point. Mitochondria were isolated from wild-type (A), ntg1-null mutant (C), or mhr1-null (E) mutant cells. After the incubation, mtDNA was extracted, and ∼10 µg of mtDNA was digested with BglII. The digests were separated by electrophoresis on a 2.0% agarose gel. The signals from the 0.8-kbp DNA fragment derived from DSBs at ori5 were normalized to those of the 1.1-kbp unit length of HS [ori5] [rho^–] mtDNA as measured by Southern blot analysis using ³²P-labeled 1.1-kbp fragments of HS [ori5] [rho^–] mtDNA as a probe. The average values from three independent experiments of the signals representing the 0.8-kbp fragments derived from DSBs at ori5, normalized to those from the unit-sized 1.1-kbp fragments of HS [ori5] [rho^–] mtDNA, are plotted. DNA was extracted from mitochondria isolated from wild-type (B), ntg1-null (D), or mhr1-null (F) cells.

ori5-containing DNA fragments extracted from isolated mitochondria are sensitive to S1 nuclease, and resistant to single-cut restriction enzymes, within the ori5-region. (A) A physical map of the locations of ori5 and the restriction sites in HS [ori5] [rho^–] mtDNA. Digestion of HS [ori5] [rho^–] mtDNA with BglII and NdeI gives rise to the ori5-containing region (760 bp) and the remainder of the DNA (380 bp). The ori5 sequence (282 bp) is indicated with an open box. (B) Ntg1-introduced nicks in the ori5-containing region of HS [ori5] [rho^–] mtDNA. HS [ori5] [rho^–] mtDNA extracted from hydrogen peroxide-untreated (lanes 1 and 2), or treated (lanes 3 and 4) mitochondria isolated from wild-type cells, was digested with BglII and NdeI. The BglII–NdeI digested DNA was treated with purified Ntg1 (1.6 nM) in the standard reaction mixture at 37°C for 30 min (lanes 2 and 4). After the removal of proteins with proteinase K, DNA digests were separated on a 2.0% denaturing (alkaline) agarose gel. The signals resulting from the ori5-containing region, the ori5-lacking region and linearized pUC119 were detected by Southern blot analysis using ³²P-labeled HS [ori5] [rho^–] mtDNA and linearized pUC119 as probes (bottom panel: Southern blot). The ratios of the signals from the ori5-containing region or the ori5-lacking region to the signal from linearized pUC119 are plotted (upper and middle panels), based on the results from three independent experiments. Error bars shown here are standard deviations. (C) Comparison of the S1 nuclease sensitivities of DNA fragments that contain or lack ori5. HS [ori5] [rho^–] mtDNA extracted from wild-type mitochondria was digested with BglII and NdeI, followed by the treatment with S1 nuclease, as indicated. DNA fragments containing ori5 (760 bp), the rest of the mtDNA (380 bp) and the smear caused by S1 nuclease were separated on a 2.0% agarose gel and detected by Southern blot analysis. (D) Quantitative analysis of DNA fragments treated with S1 nuclease. The values of the signals derived from DNA fragments not treated with S1 nuclease were defined as 1. The relative values of signals derived from undegraded DNA fragments, which contain (filled triangles) or lack (open triangles) the ori5 region are plotted. (E) Resistance of HS [ori5] [rho^–] mtDNA to single-cut enzymes within or adjacent to the ori5-region. HS [ori5] [rho^–] mtDNA from wild-type cells digested with BglII, and ori5-containing DNA fragments amplified by PCR, were treated (or not) with MnlI, HphI, EcoRV or NdeI and then separated on a 2.0% agarose gel. Marker, 100-bp ladder. (F) Removal of oxidized 5-OHU bases located at the middle of the 3′-DIG labeled 30-mer oligos by purified Ntg1. The 3′-DIG-labeled 30-mer oligos containing thymine base instead of 5-OHU were used as a control. The 30-mer oligos harboring 5-OHU or no damage were incubated with indicated amounts of Ntg1 (left panel) or C▵ntg1 (right panel) for 30 min at 37°C. The oligos in the reactions were separated by electrophoresis on a 20% polyacrylamide-urea gel. The signals representing the 3′-DIG-labeled oligos were detected using anti-DIG AP conjugates.

Increases in mtDNA copy number are triggered by ROS. When the levels of ROS increase, mtDNA is oxidatively modified at active replication origins such as ori5. These modifications, which accumulate in DNA structures with more single-stranded character, are recognized by Ntg1, which introduces a DSB at the site. The increased levels of DSBs at replication origins enhance Mhr1-mediated initiation of rolling-circle mtDNA replication, which results in an increase in mtDNA copy number.