@phdthesis{oai:sucra.repo.nii.ac.jp:00018678,
 author = {ELLIOT C. BRADSHAW},
 month = {},
 note = {92 p., The mitochondrial genome is a small unit residing within the mitochondrial matrix  that  encodes respiratory  chain  subunits, tRNAs and rRNAs essential for the majority of ATP production within most eukaryotic cells. Loss of mitochondrial genomic integrity by either point- or deletion-mutagenesis can lead to respiratory defects and several diseases. Mitochondrial DNA （mtDNA） exists as multiple copies within each cell, allowing for complementation of mutant mtDNA with wild-type copies. A mixture of wild-type and mutant molecules, termed heteroplasmy, can lead to mitochondrial dysfunction and disease if the proportion of mutant mtDNA molecules becomes sufficiently high. Deleted mtDNA molecules, which can be several kilobases shorter than wild-type, are particularly hazardous due to the replicative advantage of relatively small molecules. Indeed, the clonal expansion of deleted mtDNA has been observed in several organisms and, in human tissues its proportion can increase with age. Therefore, understanding the mechanisms that govern the generation and clonal expansion of deleted mtDNA may shed light on the aging process and give insights into treatments for mitochondrial diseases.
 In this study, we report that ribonucleotide reductase （RNR）, which catalyzes the rate-limiting step of deoxyribonucleotide triphosphate （dNTP） synthesis, regulates the replicative advantage of small mtDNA in heteroplasmic yeast cells. Crosses of parental yeast cells containing wild-type （ρ⁺） mtDNA with respiratory-deficient （ρ⁻） cells containing small mtDNA, results in production of diploid cells containing a heteroplasmic mixture of both parental mtDNA alleles. Due to the replicative advantage of small mtDNA, the majority of colonies formed  from  these crosses lack mitochondrial function, as demonstrated by an inability to grow in non-fermentable media. SML1 encodes a protein inhibitor of RNR, and RNR1 encodes the large subunit of RNR. Deletion of SML1  or  overexpression  of  RNR1 significantly increases the proportion of ρ⁺ colonies during heteroplasmy with small mtDNA. Reducing RNR activity by overexpressing SML1 produces the opposite effect. In addition, using Ex Taq and KOD Dash polymerases, we observe a replicative advantage for small template DNA over large in vitro, but only at low dNTP concentrations. These results indicate that dNTP insufficiency contributes to the replicative advantage of small mtDNA over wild-type in yeast and cytosolic dNTP synthesis by RNR is an important regulator of heteroplasmy involving small mtDNA molecules.
 Over 100 genes contribute to mtDNA  stability  in  S.  cerevisiae  and  genetic requirements for stable mtDNA maintenance depend on growth conditions. In order to study deletion mutagenesis, we constructed a double-mutant strain that undergoes rapid loss of respiratory function. ABF2 encodes a histone-like mtDNA binding protein that is required for mtDNA stability in fermentable media. MHR1 encodes an mtDNA recombinase that promotes homologous pairing, a key step in recombination-mediated repair and in the initiation of rolling-circle mtDNA replication. Deletion of MHR1 causes loss of mtDNA regardless of carbon source, however the mhr1-1 mutation allows for mtDNA maintenance at a permissive temperature but displays significantly impaired recombination function. We show that Δabf2 mhr1-1 double-mutant cells selectively grown in non-fermentable media are ρ⁺, but rapidly lose respiratory function when shifted to fermentable media due to mtDNA deletion mutagenesis. Importantly, exogenous MHR1 overexpression significantly rescues this mtDNA-loss phenotype, suggesting that Mhr1-driven mtDNA replication and homologous recombination are crucial for prevention of mtDNA deletion mutagenesis.
 Glucose is the preferred carbon source for yeast cells, and its depletion or substitution with nonfermentable carbon sources results in a vastly different transcriptional landscape. One major consequence of glucose depletion is an immediate drop in cytosolic pH, which acts as a second messenger for glucose. We explored whether depletion of glucose during the vegetative growth of diploid cells leads to changes in heteroplasmy of wild-type and small mtDNA. We found that acidification of the cytosol promotes formation of ρ⁺ colonies and increases wild-type mtDNA content over several generations, while high glucose media or alkaline cytosolic pH suppress this effect. We confirmed that this mitochondrial genetic response to cytosolic acidification occurs independently of the general stress response by MSN2/4, through an as of yet undetermined mechanism.
 Together, this study introduces two regulatory mechanisms that influence the proportional amount of deleted mtDNA over wild-type in yeast and demonstrates that cytosolic pH influences the rate of ρ⁺ colony formation from heteroplasmic cells containing ρ⁺ and ρ⁻ mtDNA. Further exploration of these topics may lead to novel interventions against mtDNA deletion-attributed mitochondrial dysfunction., Chapter 1: Introduction
1.1   The diverse metabolic functions of mitochondria ・・・・・・・・11
1.2   Mitochondrial quality control ・・・・・・・・・・・・・・12
1.3   Mitochondrial DNA metabolism・・・・・・・・・・・・・・15
1.3.1   Replication of mammalian mtDNA ・・・・・・・・・・・15
1.3.2   RNA-primed mtDNA replication in Saccharomyces cerevisiae ・・・16
1.3.3   Recombination-driven mtDNA replication ・・・・・・・・・17
1.4   MtDNA deletion mutations ・・・・・・・・・・・・・・・19
1.5   Aims of this study ・・・・・・・・・・・・・・・・・・20
1.6   Figures ・・・・・・・・・・・・・・・・・・・・・21
Figure 1.1   Models of mammalian mtDNA replication ・・・・・・・21
Figure 1.2   Model of rapid mtDNA segregation from heteroplasmy to homoplasmy in S. cerevisiae ・・・・・・・・・・・・22
Chapter 2:   Regulation of small mitochondrial DNA replicative advantage by ribonucleotide reductase in Saccharomyces cerevisiae
2.1   Introduction ・・・・・・・・・・・・・・・・・・・・23
2.1.1   Ribonucleotide reductase・・・・・・・・・・・・・・23
2.1.2   The Mec1/Rad53 DNA damage checkpoint pathway ・・・・・・23
2.1.3   Suppressive mtDNA ・・・・・・・・・・・・・・・・24
2.2   Materials and methods ・・・・・・・・・・・・・・・・25
2.2.1   Yeast strains and transformation ・・・・・・・・・・・・25
2.2.2   Yeast crossing experiments ・・・・・・・・・・・・・・25
2.2.3   Quantification of mtDNA levels in heteroplasmic cells ・・・・・25
2.2.4   Western blotting・・・・・・・・・・・・・・・・・26
2.2.5   PCR assay for competitive template amplification under varying dNTP concentrations ・・・・・・・・・・・・・・・・・26
2.2.6   Microscopy ・・・・・・・・・・・・・・・・・・・27
2.3   Results ・・・・・・・・・・・・・・・・・・・・・27
2.3.1   Sml1 is required for the hypersuppressive phenotype ・・・・・27
2.3.2   RNR1 overexpression enhances ρ⁺ mtDNA replication in  hypersuppressive crosses・・・・・・・・・・・・・・29
2.3.3   Overproducing Sml1 in Δsml1 cells restores the hypersuppressive phenotype ・・・・・・・・・・・・・・・・・・・29
2.3.4   GND1 overexpression increases ρ⁺ colony formation in hypersuppressive crosses ・・・・・・・・・・・・・・30
2.3.5   Low dNTP concentration enhances the replicative advantage of small template DNA over large in vitro ・・・・・・・・・・・・31
2.4   Discussion・・・・・・・・・・・・・・・・・・・・32
2.5   Figures and table ・・・・・・・・・・・・・・・・・・34
Figure 2.1   SML1 deletion increases the proportional amounts of ρ⁺ colonies  and full-length mtDNA during heteroplasmy with hypersuppressive mtDNA  ・・・・・・・・・・・・・・・・・・34
Figure 2.2   Effect of SML1 deletion on heteroplasmic cells containing different HS ρ⁻ and normal suppressive ρ⁻ alleles ・・・・・・・・36
Figure 2.3   RNR1 overexpression increases respiratory function and ρ⁺ mtDNA content in heteroplasmic cells・・・・・・・・・・・37
Figure 2.4   SML1 overexpression restores the hypersuppressive phenotype in Δsml1 cells  ・・・・・・・・・・・・・・・・・38
Figure 2.5   Overexpression of genes encoding the NADPH producing enzymes  of the pentose phosphate pathway・・・・・・・・・・39
Figure 2.6   Competitive amplification of DNA templates of different lengths over a range of dNTP concentrations in vitro ・・・・・・・40
Figure 2.7   Model for the role of cytosolic dNTP synthesis in regulating the replicative advantage of small mtDNA during heteroplasmy in yeast・・・・・・・・・・・・・・・・・・・・・41
Table 2.1    Yeast strains used in this chapter ・・・・・・・・・・42
Chapter 3: Prevention of mitochondrial genomic instability in Saccharomyces cerevisiae by the mitochondrial recombinase Mhr1
3.1   Introduction ・・・・・・・・・・・・・・・・・・・・44
3.1.1   The mitochondrial nucleoid protein Abf2 ・・・・・・・・・44
3.1.2   Functional roles of the mtDNA recombinase Mhr1・・・・・・44
3.2   Materials and methods ・・・・・・・・・・・・・・・・45
3.2.1   Yeast strains and media・・・・・・・・・・・・・・・45
3.2.2   Mitochondrial nucleoid analysis・・・・・・・・・・・・46
3.2.3   Tetrad analysis・・・・・・・・・・・・・・・・・・46
3.2.4   Purification of yeast mtDNA and analysis by restriction digestion・・46
3.2.5   Southern blot analysis ・・・・・・・・・・・・・・・46
3.2.5   Analysis of mtDNA level by quantitative real‐time PCR ・・・・・47
3.3   Results ・・・・・・・・・・・・・・・・・・・・・47
3.3.1   Double-mutant Δabf2 mhr1-1 cells rapidly lose respiratory function in fermentable media・・・・・・・・・・・・・・・・47
3.3.2   Nucleoid numbers are significantly reduced in Δabf2 mhr1-1 cells・・・・48
3.3.3   Loss of MHR1 causes mtDNA fragmentation ・・・・・・・・49
3.3.4   MtDNA deletion mutagenesis in Δabf2 mhr1‐1 cells ・・・・・・49
3.3.5   Mhr1 overproduction prevents mtDNA deletion mutagenesis・・・51
3.4   Discussion・・・・・・・・・・・・・・・・・・・・53
3.5   Figures and table ・・・・・・・・・・・・・・・・・・55
Figure 3.1   Respiratory function loss in Δabf2, mhr1-1 or Δabf2 mhr1-1 cells ..55
Figure 3.2   Mitochondrial nucleoid signals in Δabf2, mhr1-1 or Δabf2 mhr1-1 cells ・・・・・・・・・・・・・・・・・・・57
Figure 3.3   Tetrad analysis of respiratory function and mtDNA deletions in Δmhr1 cells ・・・・・・・・・・・・・・・・・58
Figure 3.4   Degree of mtDNA suppressivity in Δabf2 or Δabf2 mhr1-1 mutant cells ・・・・・・・・・・・・・・・・・・・59
Figure 3.5   Southern blot analysis of ApaI‐digested mtDNA from Δabf2 or Δabf2 mhr1-1 cells ・・・・・・・・・・・・・・・・・60
Figure 3.6   Effects of Mhr1 overproduction on mtDNA content and respiratory function・・・・・・・・・・・・・・・・・・61
Figure 3.7   Model for the prevention of mtDNA deletion mutagenesis by Mhr1-driven recombination and mtDNA replication ・・・・・・63
Table 3.1    Yeast strains used in this chapter ・・・・・・・・・・64
Chapter 4: Influence of cytosolic pH on small mitochondrial DNA heteroplasmy in Saccharomyces cerevisiae
4.1   Introduction ・・・・・・・・・・・・・・・・・・・・65
4.2   Materials and methods ・・・・・・・・・・・・・・・・65
4.2.1   Yeast crossing experiments ・・・・・・・・・・・・・・65
4.2.2   Detection of cytosolic pH with superecliptic pHluorin ・・・・・66
4.2.3   Quantification of mtDNA levels in heteroplasmic cells  ・・・・・66
4.3   Results ・・・・・・・・・・・・・・・・・・・・・67
4.3.1   Vegetative growth in low glucose media promotes ρ⁺ colony formation from heteroplasmic cells with hypersuppressive mtDNA ・・・・67
4.3.2   An early drop in cytosolic pH stimulates ρ⁺ colony formation ・・・67
4.3.3   Increased cytosolic pH suppresses ρ⁺ colony formation and ρ⁺ mtDNA level・・・・・・・・・・・・・・・・・・・・・68
4.3.4   The general stress response transcription factors Msn2 and Msn4 are not required for ρ⁺ colony formation in low pH media ・・・・・・69
4.4   Discussion・・・・・・・・・・・・・・・・・・・・69
4.5   Figures and table ・・・・・・・・・・・・・・・・・・71
Figure 4.1   Influence of glucose concentration on heteroplasmy of wild-type and small mtDNA over several generations of vegetative growth ・・・・・・71
Figure 4.2   Effect of cytosolic pH on ρ⁺ colony formation during vegetative growth of heteroplasmic cells ・・・・・・・・・・・72
Figure 4.3   Effect of buffered media on cytosolic acidification-driven ρ⁺ colony formation and relative amounts of ρ⁺ and HS ρ⁻mtDNA in cultures・・・・・・・・・・・・・・・・・・・・・74
Figure 4.4   Analysis of the involvement of the transcription factors Msn2 and Msn4 in formation of ρ⁺ colonies during vegetative growth of heteroplasmic cells ・・・・・・・・・・・・・・・76
Figure 4.5   Model of the effect of cytosolic pH on the vegetative segregation of mtDNA alleles ・・・・・・・・・・・・・・・・77
Table 4.1   Yeast strains used in this chapter ・・・・・・・・・77
Chapter 5: Conclusion
5.1   Concluding remarks・・・・・・・・・・・・・・・・・78
References
References・・・・・・・・・・・・・・・・・・・・・・79, 指導教員 : 吉田稔, text, application/pdf},
 school = {埼玉大学},
 title = {Regulation of deleted mtDNA heteroplasmy in Saccharomyces cerevisiae},
 year = {2018},
 yomi = {エリオット コルビ ブラッドシャウ}
}