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Din7 and Mhr1 expression levels regulate double-strand-break-induced replication and recombination of mtDNA at ori5 in yeast.

Ling F, Hori A, Yoshitani A, Niu R, Yoshida M, Shibata T - Nucleic Acids Res. (2013)

Bottom Line: Although replication and recombination profoundly influence mitochondrial inheritance, the regulatory mechanisms that determine the choice between these pathways remain unknown.However, simultaneous overproduction of Mhr1 suppressed all of these phenotypes and enhanced homologous recombination.Our results suggest that after homologous pairing, the relative activity levels of Din7 and Mhr1 modulate the preference for replication versus homologous recombination to repair DSBs at ori5.

View Article: PubMed Central - PubMed

Affiliation: Chemical Genetics Laboratory, RIKEN, Hirosawa 2-1, Wako-shi, Saitama 351-0198, Japan. ling@postman.riken.go.jp

ABSTRACT
The Ntg1 and Mhr1 proteins initiate rolling-circle mitochondrial (mt) DNA replication to achieve homoplasmy, and they also induce homologous recombination to maintain mitochondrial genome integrity. Although replication and recombination profoundly influence mitochondrial inheritance, the regulatory mechanisms that determine the choice between these pathways remain unknown. In Saccharomyces cerevisiae, double-strand breaks (DSBs) introduced by Ntg1 at the mitochondrial replication origin ori5 induce homologous DNA pairing by Mhr1, and reactive oxygen species (ROS) enhance production of DSBs. Here, we show that a mitochondrial nuclease encoded by the nuclear gene DIN7 (DNA damage inducible gene) has 5'-exodeoxyribonuclease activity. Using a small ρ(-) mtDNA bearing ori5 (hypersuppressive; HS) as a model mtDNA, we revealed that DIN7 is required for ROS-enhanced mtDNA replication and recombination that are both induced at ori5. Din7 overproduction enhanced Mhr1-dependent mtDNA replication and increased the number of residual DSBs at ori5 in HS-ρ(-) cells and increased deletion mutagenesis at the ori5 region in ρ(+) cells. However, simultaneous overproduction of Mhr1 suppressed all of these phenotypes and enhanced homologous recombination. Our results suggest that after homologous pairing, the relative activity levels of Din7 and Mhr1 modulate the preference for replication versus homologous recombination to repair DSBs at ori5.

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Analysis of DSBs at ori5 and mtDNA copy number in Δdin7 and Δmhr1 mutant cells. (A) Physical map of the ori5 and DSB sites at ori5 in HSC-1, the HS ρ− mtDNA used in this study. The ori5 sequence is framed with an open box. The DSB site is indicated by an arrow. (B) Example of a gel profile, showing an analysis of the DSB at ori5 and mtDNA copy number. The wild-type and indicated mutant cells containing the HS ρ− mtDNA were grown to log phase in YPD medium and then divided into two samples. One sample was resuspended in PBS, whereas the other was treated with 10 µM hydrogen peroxide for 1 h at 30°C. Whole cellular DNA (∼10 µg) isolated from wild-type and mutant yeast cells was digested with BglII, separated by electrophoresis on a 2% agarose gel and transferred to Hybond-N+ membranes (GE Healthcare). The HS ρ− mtDNA on the membranes was detected by Southern hybridization using 32P-labeled HS ρ− mtDNA and the NUC1 gene as probes, as described previously (5). WT, wild-type; NUC1, 3.2-kb DNA fragment containing 0.99-kb NUC1 gene; 1.1-kb, unit size of BglII-digested HS ρ− mtDNA (1.1 kb); 0.8-kb (DSB) and 0.8-kb DNA fragment derived from the unique DSB at ori5 by the BglII-digest; asterisks indicates mtDNA containing single-stranded regions resistant to BglII digestion. (C) Quantitative representation of the DSBs at ori5 and mtDNA copy numbers. The amount of DSBs at ori5 in HS ρ− mtDNA in the indicated cells was calculated based on the signals from the 0.8-kb DNA fragment, generated only when a DSB is present, relative to the signals from the unit-sized 1.1-kb DNA of HS ρ− mtDNA. MtDNA copy numbers were expressed based on the signals from unit-sized 1.1-kb HS ρ− mtDNA relative to those from the 3.2-kb DNA fragment containing the NUC1 gene. Both signals were normalized against those of HS ρ− mtDNA isolated from the wild-type cells without hydrogen peroxide treatment and plotted. Each bar represents the results of at least two independent experiments. (D) Din7 expression level in hydrogen peroxide-treated cells. Wild-type cells containing HS ρ– mtDNA were grown to log phase in 2 l of YPD medium and then divided into two samples. One sample was resuspended in PBS, and the other was treated with 10 µM hydrogen peroxide for 1 h at 30°C. Then, mitochondria were isolated, and mitochondrial extracts were prepared for immunoblot analysis using an anti-Din7 rabbit serum prepared for this study. Note: to detect optimal signals for the mitochondrial outer-membrane protein porin, the prepared mitochondrial extracts were diluted 100-fold before being used in immunoblot analysis using a monoclonal anti-porin antibody.
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gkt273-F2: Analysis of DSBs at ori5 and mtDNA copy number in Δdin7 and Δmhr1 mutant cells. (A) Physical map of the ori5 and DSB sites at ori5 in HSC-1, the HS ρ− mtDNA used in this study. The ori5 sequence is framed with an open box. The DSB site is indicated by an arrow. (B) Example of a gel profile, showing an analysis of the DSB at ori5 and mtDNA copy number. The wild-type and indicated mutant cells containing the HS ρ− mtDNA were grown to log phase in YPD medium and then divided into two samples. One sample was resuspended in PBS, whereas the other was treated with 10 µM hydrogen peroxide for 1 h at 30°C. Whole cellular DNA (∼10 µg) isolated from wild-type and mutant yeast cells was digested with BglII, separated by electrophoresis on a 2% agarose gel and transferred to Hybond-N+ membranes (GE Healthcare). The HS ρ− mtDNA on the membranes was detected by Southern hybridization using 32P-labeled HS ρ− mtDNA and the NUC1 gene as probes, as described previously (5). WT, wild-type; NUC1, 3.2-kb DNA fragment containing 0.99-kb NUC1 gene; 1.1-kb, unit size of BglII-digested HS ρ− mtDNA (1.1 kb); 0.8-kb (DSB) and 0.8-kb DNA fragment derived from the unique DSB at ori5 by the BglII-digest; asterisks indicates mtDNA containing single-stranded regions resistant to BglII digestion. (C) Quantitative representation of the DSBs at ori5 and mtDNA copy numbers. The amount of DSBs at ori5 in HS ρ− mtDNA in the indicated cells was calculated based on the signals from the 0.8-kb DNA fragment, generated only when a DSB is present, relative to the signals from the unit-sized 1.1-kb DNA of HS ρ− mtDNA. MtDNA copy numbers were expressed based on the signals from unit-sized 1.1-kb HS ρ− mtDNA relative to those from the 3.2-kb DNA fragment containing the NUC1 gene. Both signals were normalized against those of HS ρ− mtDNA isolated from the wild-type cells without hydrogen peroxide treatment and plotted. Each bar represents the results of at least two independent experiments. (D) Din7 expression level in hydrogen peroxide-treated cells. Wild-type cells containing HS ρ– mtDNA were grown to log phase in 2 l of YPD medium and then divided into two samples. One sample was resuspended in PBS, and the other was treated with 10 µM hydrogen peroxide for 1 h at 30°C. Then, mitochondria were isolated, and mitochondrial extracts were prepared for immunoblot analysis using an anti-Din7 rabbit serum prepared for this study. Note: to detect optimal signals for the mitochondrial outer-membrane protein porin, the prepared mitochondrial extracts were diluted 100-fold before being used in immunoblot analysis using a monoclonal anti-porin antibody.

Mentions: Although overexpressing Din7 increased petite formation and DIN7 disruption did not alter mtDNA stability (19), we observed a slight deficiency in Mhr1-dependent polar recombination: i.e. we observed a decrease in the transmission ratio of the ω+-linked chloramphenicol-resistant marker, from 98% in the presence of DIN7 to 81% in its absence [F.L., unpublished manuscript and see (8)]. To investigate the role of Din7 in repairing DSBs at ori5 and regulating mtDNA copy number, we used wild-type, Δdin7, Δmhr1 and Δdin7Δmhr1 cells that contained HS ρ− mtDNA (HSC-1; Table 1). The HSC-1 ρ− mtDNA is a 1.1-kb mtDNA segment containing a replication origin, ori5 (DDB/EMBL/GenBank accession number AB182994). For these experiments, whole cellular DNA was prepared, treated with restriction endonuclease BglII and then subjected to gel electrophoresis. Next, mtDNA and nuclear DNA were detected by Southern hybridization using the 1.1-kb HSC-1 ρ− mtDNA and a fragment of the NUC1 0.99-kb ORF as probes, as previously described (5,6). The amount of DSBs in each sample was calculated based on the ratio between the signal of a 0.8-kb DNA fragment that is obtained in BglII-digested DNA only when a DSB is present at ori5 and the signal of a larger fragment that is always present in BglII-digested DNA (unit size 1.1-kb HSC-1 ρ− mtDNA, Figure 2A). The amount of mtDNA (i.e. mtDNA copy number) in each sample was expressed as the ratio of the signal of the 1.1-kb ρ− mtDNA to the signal from a BglII digest-derived 3.2-kb DNA fragment containing the NUC1 gene.Figure 2.


Din7 and Mhr1 expression levels regulate double-strand-break-induced replication and recombination of mtDNA at ori5 in yeast.

Ling F, Hori A, Yoshitani A, Niu R, Yoshida M, Shibata T - Nucleic Acids Res. (2013)

Analysis of DSBs at ori5 and mtDNA copy number in Δdin7 and Δmhr1 mutant cells. (A) Physical map of the ori5 and DSB sites at ori5 in HSC-1, the HS ρ− mtDNA used in this study. The ori5 sequence is framed with an open box. The DSB site is indicated by an arrow. (B) Example of a gel profile, showing an analysis of the DSB at ori5 and mtDNA copy number. The wild-type and indicated mutant cells containing the HS ρ− mtDNA were grown to log phase in YPD medium and then divided into two samples. One sample was resuspended in PBS, whereas the other was treated with 10 µM hydrogen peroxide for 1 h at 30°C. Whole cellular DNA (∼10 µg) isolated from wild-type and mutant yeast cells was digested with BglII, separated by electrophoresis on a 2% agarose gel and transferred to Hybond-N+ membranes (GE Healthcare). The HS ρ− mtDNA on the membranes was detected by Southern hybridization using 32P-labeled HS ρ− mtDNA and the NUC1 gene as probes, as described previously (5). WT, wild-type; NUC1, 3.2-kb DNA fragment containing 0.99-kb NUC1 gene; 1.1-kb, unit size of BglII-digested HS ρ− mtDNA (1.1 kb); 0.8-kb (DSB) and 0.8-kb DNA fragment derived from the unique DSB at ori5 by the BglII-digest; asterisks indicates mtDNA containing single-stranded regions resistant to BglII digestion. (C) Quantitative representation of the DSBs at ori5 and mtDNA copy numbers. The amount of DSBs at ori5 in HS ρ− mtDNA in the indicated cells was calculated based on the signals from the 0.8-kb DNA fragment, generated only when a DSB is present, relative to the signals from the unit-sized 1.1-kb DNA of HS ρ− mtDNA. MtDNA copy numbers were expressed based on the signals from unit-sized 1.1-kb HS ρ− mtDNA relative to those from the 3.2-kb DNA fragment containing the NUC1 gene. Both signals were normalized against those of HS ρ− mtDNA isolated from the wild-type cells without hydrogen peroxide treatment and plotted. Each bar represents the results of at least two independent experiments. (D) Din7 expression level in hydrogen peroxide-treated cells. Wild-type cells containing HS ρ– mtDNA were grown to log phase in 2 l of YPD medium and then divided into two samples. One sample was resuspended in PBS, and the other was treated with 10 µM hydrogen peroxide for 1 h at 30°C. Then, mitochondria were isolated, and mitochondrial extracts were prepared for immunoblot analysis using an anti-Din7 rabbit serum prepared for this study. Note: to detect optimal signals for the mitochondrial outer-membrane protein porin, the prepared mitochondrial extracts were diluted 100-fold before being used in immunoblot analysis using a monoclonal anti-porin antibody.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3675488&req=5

gkt273-F2: Analysis of DSBs at ori5 and mtDNA copy number in Δdin7 and Δmhr1 mutant cells. (A) Physical map of the ori5 and DSB sites at ori5 in HSC-1, the HS ρ− mtDNA used in this study. The ori5 sequence is framed with an open box. The DSB site is indicated by an arrow. (B) Example of a gel profile, showing an analysis of the DSB at ori5 and mtDNA copy number. The wild-type and indicated mutant cells containing the HS ρ− mtDNA were grown to log phase in YPD medium and then divided into two samples. One sample was resuspended in PBS, whereas the other was treated with 10 µM hydrogen peroxide for 1 h at 30°C. Whole cellular DNA (∼10 µg) isolated from wild-type and mutant yeast cells was digested with BglII, separated by electrophoresis on a 2% agarose gel and transferred to Hybond-N+ membranes (GE Healthcare). The HS ρ− mtDNA on the membranes was detected by Southern hybridization using 32P-labeled HS ρ− mtDNA and the NUC1 gene as probes, as described previously (5). WT, wild-type; NUC1, 3.2-kb DNA fragment containing 0.99-kb NUC1 gene; 1.1-kb, unit size of BglII-digested HS ρ− mtDNA (1.1 kb); 0.8-kb (DSB) and 0.8-kb DNA fragment derived from the unique DSB at ori5 by the BglII-digest; asterisks indicates mtDNA containing single-stranded regions resistant to BglII digestion. (C) Quantitative representation of the DSBs at ori5 and mtDNA copy numbers. The amount of DSBs at ori5 in HS ρ− mtDNA in the indicated cells was calculated based on the signals from the 0.8-kb DNA fragment, generated only when a DSB is present, relative to the signals from the unit-sized 1.1-kb DNA of HS ρ− mtDNA. MtDNA copy numbers were expressed based on the signals from unit-sized 1.1-kb HS ρ− mtDNA relative to those from the 3.2-kb DNA fragment containing the NUC1 gene. Both signals were normalized against those of HS ρ− mtDNA isolated from the wild-type cells without hydrogen peroxide treatment and plotted. Each bar represents the results of at least two independent experiments. (D) Din7 expression level in hydrogen peroxide-treated cells. Wild-type cells containing HS ρ– mtDNA were grown to log phase in 2 l of YPD medium and then divided into two samples. One sample was resuspended in PBS, and the other was treated with 10 µM hydrogen peroxide for 1 h at 30°C. Then, mitochondria were isolated, and mitochondrial extracts were prepared for immunoblot analysis using an anti-Din7 rabbit serum prepared for this study. Note: to detect optimal signals for the mitochondrial outer-membrane protein porin, the prepared mitochondrial extracts were diluted 100-fold before being used in immunoblot analysis using a monoclonal anti-porin antibody.
Mentions: Although overexpressing Din7 increased petite formation and DIN7 disruption did not alter mtDNA stability (19), we observed a slight deficiency in Mhr1-dependent polar recombination: i.e. we observed a decrease in the transmission ratio of the ω+-linked chloramphenicol-resistant marker, from 98% in the presence of DIN7 to 81% in its absence [F.L., unpublished manuscript and see (8)]. To investigate the role of Din7 in repairing DSBs at ori5 and regulating mtDNA copy number, we used wild-type, Δdin7, Δmhr1 and Δdin7Δmhr1 cells that contained HS ρ− mtDNA (HSC-1; Table 1). The HSC-1 ρ− mtDNA is a 1.1-kb mtDNA segment containing a replication origin, ori5 (DDB/EMBL/GenBank accession number AB182994). For these experiments, whole cellular DNA was prepared, treated with restriction endonuclease BglII and then subjected to gel electrophoresis. Next, mtDNA and nuclear DNA were detected by Southern hybridization using the 1.1-kb HSC-1 ρ− mtDNA and a fragment of the NUC1 0.99-kb ORF as probes, as previously described (5,6). The amount of DSBs in each sample was calculated based on the ratio between the signal of a 0.8-kb DNA fragment that is obtained in BglII-digested DNA only when a DSB is present at ori5 and the signal of a larger fragment that is always present in BglII-digested DNA (unit size 1.1-kb HSC-1 ρ− mtDNA, Figure 2A). The amount of mtDNA (i.e. mtDNA copy number) in each sample was expressed as the ratio of the signal of the 1.1-kb ρ− mtDNA to the signal from a BglII digest-derived 3.2-kb DNA fragment containing the NUC1 gene.Figure 2.

Bottom Line: Although replication and recombination profoundly influence mitochondrial inheritance, the regulatory mechanisms that determine the choice between these pathways remain unknown.However, simultaneous overproduction of Mhr1 suppressed all of these phenotypes and enhanced homologous recombination.Our results suggest that after homologous pairing, the relative activity levels of Din7 and Mhr1 modulate the preference for replication versus homologous recombination to repair DSBs at ori5.

View Article: PubMed Central - PubMed

Affiliation: Chemical Genetics Laboratory, RIKEN, Hirosawa 2-1, Wako-shi, Saitama 351-0198, Japan. ling@postman.riken.go.jp

ABSTRACT
The Ntg1 and Mhr1 proteins initiate rolling-circle mitochondrial (mt) DNA replication to achieve homoplasmy, and they also induce homologous recombination to maintain mitochondrial genome integrity. Although replication and recombination profoundly influence mitochondrial inheritance, the regulatory mechanisms that determine the choice between these pathways remain unknown. In Saccharomyces cerevisiae, double-strand breaks (DSBs) introduced by Ntg1 at the mitochondrial replication origin ori5 induce homologous DNA pairing by Mhr1, and reactive oxygen species (ROS) enhance production of DSBs. Here, we show that a mitochondrial nuclease encoded by the nuclear gene DIN7 (DNA damage inducible gene) has 5'-exodeoxyribonuclease activity. Using a small ρ(-) mtDNA bearing ori5 (hypersuppressive; HS) as a model mtDNA, we revealed that DIN7 is required for ROS-enhanced mtDNA replication and recombination that are both induced at ori5. Din7 overproduction enhanced Mhr1-dependent mtDNA replication and increased the number of residual DSBs at ori5 in HS-ρ(-) cells and increased deletion mutagenesis at the ori5 region in ρ(+) cells. However, simultaneous overproduction of Mhr1 suppressed all of these phenotypes and enhanced homologous recombination. Our results suggest that after homologous pairing, the relative activity levels of Din7 and Mhr1 modulate the preference for replication versus homologous recombination to repair DSBs at ori5.

Show MeSH
Related in: MedlinePlus