Feng Ling - Academia.edu (original) (raw)
Papers by Feng Ling
number in isolated yeast mitochondria by triggering recombination-mediated replication
G3 (Bethesda, Md.), Sep 7, 2017
Small mitochondrial genomes can behave as selfish elements by displacing wild-type genomes regard... more Small mitochondrial genomes can behave as selfish elements by displacing wild-type genomes regardless of their detriment to the host organism. In the budding yeast Saccharomyces cerevisiae, small hypersuppressive mtDNA transiently coexist with wild-type in a state of heteroplasmy, wherein the replicative advantage of the small mtDNA outcompetes wild-type and produces offspring without respiratory capacity in >95% of colonies. The cytosolic enzyme ribonucleotide reductase (RNR) catalyzes the rate-limiting step in dNTP synthesis and its inhibition has been correlated with increased petite colony formation, reflecting loss of respiratory function. Here, we used heteroplasmic diploids containing wild-type (rho(+)) and suppressive (rho(-)) or hypersuppressive (HS rho(-)) mitochondrial genomes to explore the effects of RNR activity on mtDNA heteroplasmy in offspring. We found that the proportion of rho(+) offspring was significantly increased by RNR overexpression or deletion of its in...
PLoS ONE, 2012
Activation of the Mec1/Rad53 damage checkpoint pathway influences mitochondrial DNA (mtDNA) conte... more Activation of the Mec1/Rad53 damage checkpoint pathway influences mitochondrial DNA (mtDNA) content and point mutagenesis in Saccharomyces cerevisiae. The effects of this conserved checkpoint pathway on mitochondrial genomes in human cells remain largely unknown. Here, we report that knockdown of the human DNA helicase RRM3 enhances phosphorylation of the cell cycle arrest kinase Chk2, indicating activation of the checkpoint via the ATM/Chk2 pathway, and increases mtDNA content independently of TFAM, a regulator of mtDNA copy number. Cell-cycle arrest did not have a consistent effect on mtDNA level: knockdown of cell cycle regulators PLK1 (polo-like kinase), MCM2, or MCM3 gave rise, respectively, to decreased, increased, or almost unchanged mtDNA levels. Therefore, we concluded that the mtDNA content increase upon RRM3 knockdown is not a response to delay of cell cycle progression. Also, we observed that RRM3 knockdown increased the levels of reactive oxygen species (ROS); two ROS scavengers, N-acetyl cysteine and vitamin C, suppressed the mtDNA content increase. On the other hand, in RRM3 knockdown cells, we detected an increase in the frequency of the common 4977-bp mtDNA deletion, a major mtDNA deletion that can be induced by abnormal ROS generation, and is associated with a decline in mitochondrial genome integrity, aging, and various mtDNA-related disorders in humans. These results suggest that increase of the mitochondrial genome by TFAM-independent mtDNA replication is connected, via oxidative stress, with the ATM/Chk2 checkpoint activation in response to DNA damage, and is accompanied by generation of the common 4977-bp deletion.
Nucleic Acids Research, 2008
Nucleic Acids Research, 2013
The Ntg1 and Mhr1 proteins initiate rolling-circle mitochondrial (mt) DNA replication to achieve ... more 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 0-exodeoxyribonuclease activity. Using a small o À 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-o À cells and increased deletion mutagenesis at the ori5 region in o + 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.
Biochemical and Biophysical Research Communications, 2008
Din7 is a DNA damage-inducible mitochondrial nuclease that modulates the stability of mitochondri... more Din7 is a DNA damage-inducible mitochondrial nuclease that modulates the stability of mitochondrial DNA (mtDNA) in Saccharomyces cerevisiae. How DIN7 gene expression is regulated, however, has remained largely unclear. Using promoter sequence alignment, we found a highly conserved 19-bp sequence in the promoter regions of DIN7 and NTG1, which encodes an oxidative stress-inducible base-excision-repair enzyme. Deletion of the 19-bp sequence markedly reduced the hydroxyurea (HU)-enhanced DIN7 promoter activity. In addition, nuclear fractions prepared from HU-treated cells were used in in vitro band shift assays to reveal the presence of currently unidentified transacting factor(s) that preferentially bound to the 19-bp region. These results suggest that the 19-bp sequence is a novel cis-acting element that is required for the regulation of DIN7 expression in response to HU-induced DNA damage.
Genes, 2020
Studies of mitochondrial (mt)DNA replication, which forms the basis of mitochondrial inheritance,... more Studies of mitochondrial (mt)DNA replication, which forms the basis of mitochondrial inheritance, have demonstrated that a rolling-circle replication mode exists in yeasts and human cells. In yeast, rolling-circle mtDNA replication mediated by homologous recombination is the predominant pathway for replication of wild-type mtDNA. In human cells, reactive oxygen species (ROS) induce rolling-circle replication to produce concatemers, linear tandem multimers linked by head-to-tail unit-sized mtDNA that promote restoration of homoplasmy from heteroplasmy. The event occurs ahead of mtDNA replication mechanisms observed in mammalian cells, especially under higher ROS load, as newly synthesized mtDNA is concatemeric in hydrogen peroxide-treated human cells. Rolling-circle replication holds promise for treatment of mtDNA heteroplasmy-attributed diseases, which are regarded as incurable. This review highlights the potential therapeutic value of rolling-circle mtDNA replication.
Bioscience, Biotechnology, and Biochemistry, 2008
Yeast DIN7 is a DNA damage-inducible gene. Its expression is increased in the absence of Dun1, a ... more Yeast DIN7 is a DNA damage-inducible gene. Its expression is increased in the absence of Dun1, a DNA damage checkpoint kinase. We identified a DIN7 promoter region responsible for Dun1-mediated downregulation and found that DIN7 expression was not further increased in response to hydroxyurea in Ádun1 cells. Thus DIN7 repression by Dun1 can be released upon DNA damage.
Genes to Cells, 2011
Mitochondrial fusion plays an important role in mitochondrial DNA (mtDNA) maintenance, although t... more Mitochondrial fusion plays an important role in mitochondrial DNA (mtDNA) maintenance, although the underlying mechanisms are unclear. In budding yeast, certain levels of reactive oxygen species (ROS) can promote recombination-mediated mtDNA replication, and mtDNA maintenance depends on the homologous DNA pairing protein Mhr1. Here, we show that the fusion of isolated yeast mitochondria, which can be monitored by the bimolecular fluorescence complementation-derived green fluorescent protein (GFP) fluorescence, increases the mtDNA copy number in a manner dependent on Mhr1. The fusion event, accompanied by the degradation of dissociated electron transport chain complex IV and transient reductions in the complex IV subunits by the inner membrane AAA proteases such as Yme1, increases ROS levels. Analysis of the initial stage of mitochondrial fusion in early log-phase cells produced similar results. Moreover, higher ROS levels in mitochondrial fusion-deficient mutant cells increased the amount of newly synthesized mtDNA, resulting in increases in the mtDNA copy number. In contrast, reducing ROS levels in yme1 null mutant cells significantly decreased the mtDNA copy number, leading to an increase in cells lacking mtDNA. Our results indicate that mitochondrial fusion induces mtDNA synthesis by facilitating ROS-triggered, recombination-mediated replication and thereby prevents the generation of mitochondria lacking DNA.
Genes, 2011
Mitochondria have their own genomic DNA. Unlike the nuclear genome, each cell contains hundreds t... more Mitochondria have their own genomic DNA. Unlike the nuclear genome, each cell contains hundreds to thousands of copies of mitochondrial DNA (mtDNA). The copies of mtDNA tend to have heterogeneous sequences, due to the high frequency of mutagenesis, but are quickly homogenized within a cell ("homoplasmy") during vegetative cell growth or through a few sexual generations. Heteroplasmy is strongly associated with mitochondrial diseases, diabetes and aging. Recent studies revealed that the yeast cell has the machinery to homogenize mtDNA, using a common DNA processing pathway with gene conversion; i.e., both genetic events are initiated by a double-stranded break, which is processed into 3' single-stranded tails. One of the tails is base-paired with the complementary sequence of the recipient double-stranded DNA to form a D-loop (homologous pairing), in which repair DNA synthesis is initiated to restore the sequence lost by the breakage. Gene conversion generates sequence diversity, depending on the divergence between the donor and recipient sequences, especially when it occurs among a number of copies of a DNA sequence family with some sequence variations, such as in
![Research paper thumbnail of DNA Recombination-Initiation Plays a Role in the Extremely Biased Inheritance of Yeast [rho-] Mitochondrial DNA That Contains the Replication Origin ori5](https://attachments.academia-assets.com/85827162/thumbnails/1.jpg)
](Molecular and Cellular Biology, 2006
Hypersuppressiveness, as observed in Saccharomyces cerevisiae, is an extremely biased inheritance... more Hypersuppressiveness, as observed in Saccharomyces cerevisiae, is an extremely biased inheritance of a small mitochondrial DNA (mtDNA) fragment that contains a replication origin (HS [rho −] mtDNA). Our previous studies showed that concatemers (linear head-to-tail multimers) are obligatory intermediates for mtDNA partitioning and are primarily formed by rolling-circle replication mediated by Mhr1, a protein required for homologous mtDNA recombination. In this study, we found that Mhr1 is required for the hypersuppressiveness of HS [ori5] [rho −] mtDNA harboring ori5, one of the replication origins of normal ([rho +]) mtDNA. In addition, we detected an Ntg1-stimulated double-strand break at the ori5 locus. Purified Ntg1, a base excision repair enzyme, introduced a double-stranded break by itself into HS [ori5] [rho −] mtDNA at ori5 isolated from yeast cells. Both hypersuppressiveness and concatemer formation of HS [ori5] [rho −] mtDNA are simultaneously suppressed by the ntg1 null mu...
Molecular biology of the cell, May 15, 2016
Mitochondria that contain a mixture of mutant and wild-type mitochondrial (mt) DNA copies are het... more Mitochondria that contain a mixture of mutant and wild-type mitochondrial (mt) DNA copies are heteroplasmic. In humans, homoplasmy is restored during early oogenesis and reprogramming of somatic cells, but the mechanism of mt-allele segregation remains unknown. In budding yeast, homoplasmy is restored by head-to-tail concatemer formation in mother cells by reactive oxygen species (ROS)-induced rolling-circle replication and selective transmission of concatemers to daughter cells, but this mechanism is not obvious in higher eukaryotes. Here, using heteroplasmic m.3243A > G primary fibroblast cells derived from MELAS patients treated with hydrogen peroxide (H2O2), we show that an optimal ROS level promotes mt-allele segregation toward wild-type and mutant mtDNA homoplasmy. Enhanced ROS level reduced the amount of intact mtDNA replication templates but increased linear tandem multimers linked by head-to-tail unit-sized mtDNA (mtDNA concatemers). ROS-triggered mt-allele segregation c...
Journal of Biological Chemistry, 2009
The abbreviations used are: dsDNA, double-stranded DNA; mtDNA, mitochondrial DNA; ssDNA, single-s... more The abbreviations used are: dsDNA, double-stranded DNA; mtDNA, mitochondrial DNA; ssDNA, single-stranded DNA; cc-dsDNA, closed circular dsDNA.
Scientific Reports
Mitochondrial (mt) DNA encodes factors essential for cellular respiration, therefore its level an... more Mitochondrial (mt) DNA encodes factors essential for cellular respiration, therefore its level and integrity are crucial. ABF2 encodes a mitochondrial DNA-binding protein and its null mutation (Δabf2) induces mtDNA instability in Saccharomyces cerevisiae. Mhr1 is a mitochondrial recombinase that mediates the predominant form of mtDNA replication and acts in mtDNA segregation and the repair of mtDNA double-stranded breaks (DSBs). However, the involvement of Mhr1 in prevention of mtDNA deletion mutagenesis is unknown. In this study we used Δabf2 mhr1-1 double-mutant cells, which lose mitochondrial function in media containing fermentable carbon sources, to investigate whether Mhr1 is a suppressor of mtDNA deletion mutagenesis. We used a suppresivity assay and Southern blot analysis to reveal that the Δabf2 mutation causes mtDNA deletions rather than an mtDNA-lacking (ρ 0) phenotype, and observed that mtDNA deletions are exacerbated by an additional mhr1-1 mutation. Loss of respiratory function due to mtDNA fragmentation occurred in ∆mhr1 and ∆abf2 mhr1-1 cells. However, exogenous introduction of Mhr1 into Δabf2 mhr1-1 cells significantly rescued respiratory growth, suggesting that Mhr1-driven homologous mtDNA recombination prevents mtDNA instability.
number in isolated yeast mitochondria by triggering recombination-mediated replication
G3 (Bethesda, Md.), Sep 7, 2017
Small mitochondrial genomes can behave as selfish elements by displacing wild-type genomes regard... more Small mitochondrial genomes can behave as selfish elements by displacing wild-type genomes regardless of their detriment to the host organism. In the budding yeast Saccharomyces cerevisiae, small hypersuppressive mtDNA transiently coexist with wild-type in a state of heteroplasmy, wherein the replicative advantage of the small mtDNA outcompetes wild-type and produces offspring without respiratory capacity in >95% of colonies. The cytosolic enzyme ribonucleotide reductase (RNR) catalyzes the rate-limiting step in dNTP synthesis and its inhibition has been correlated with increased petite colony formation, reflecting loss of respiratory function. Here, we used heteroplasmic diploids containing wild-type (rho(+)) and suppressive (rho(-)) or hypersuppressive (HS rho(-)) mitochondrial genomes to explore the effects of RNR activity on mtDNA heteroplasmy in offspring. We found that the proportion of rho(+) offspring was significantly increased by RNR overexpression or deletion of its in...
PLoS ONE, 2012
Activation of the Mec1/Rad53 damage checkpoint pathway influences mitochondrial DNA (mtDNA) conte... more Activation of the Mec1/Rad53 damage checkpoint pathway influences mitochondrial DNA (mtDNA) content and point mutagenesis in Saccharomyces cerevisiae. The effects of this conserved checkpoint pathway on mitochondrial genomes in human cells remain largely unknown. Here, we report that knockdown of the human DNA helicase RRM3 enhances phosphorylation of the cell cycle arrest kinase Chk2, indicating activation of the checkpoint via the ATM/Chk2 pathway, and increases mtDNA content independently of TFAM, a regulator of mtDNA copy number. Cell-cycle arrest did not have a consistent effect on mtDNA level: knockdown of cell cycle regulators PLK1 (polo-like kinase), MCM2, or MCM3 gave rise, respectively, to decreased, increased, or almost unchanged mtDNA levels. Therefore, we concluded that the mtDNA content increase upon RRM3 knockdown is not a response to delay of cell cycle progression. Also, we observed that RRM3 knockdown increased the levels of reactive oxygen species (ROS); two ROS scavengers, N-acetyl cysteine and vitamin C, suppressed the mtDNA content increase. On the other hand, in RRM3 knockdown cells, we detected an increase in the frequency of the common 4977-bp mtDNA deletion, a major mtDNA deletion that can be induced by abnormal ROS generation, and is associated with a decline in mitochondrial genome integrity, aging, and various mtDNA-related disorders in humans. These results suggest that increase of the mitochondrial genome by TFAM-independent mtDNA replication is connected, via oxidative stress, with the ATM/Chk2 checkpoint activation in response to DNA damage, and is accompanied by generation of the common 4977-bp deletion.
Nucleic Acids Research, 2008
Nucleic Acids Research, 2013
The Ntg1 and Mhr1 proteins initiate rolling-circle mitochondrial (mt) DNA replication to achieve ... more 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 0-exodeoxyribonuclease activity. Using a small o À 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-o À cells and increased deletion mutagenesis at the ori5 region in o + 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.
Biochemical and Biophysical Research Communications, 2008
Din7 is a DNA damage-inducible mitochondrial nuclease that modulates the stability of mitochondri... more Din7 is a DNA damage-inducible mitochondrial nuclease that modulates the stability of mitochondrial DNA (mtDNA) in Saccharomyces cerevisiae. How DIN7 gene expression is regulated, however, has remained largely unclear. Using promoter sequence alignment, we found a highly conserved 19-bp sequence in the promoter regions of DIN7 and NTG1, which encodes an oxidative stress-inducible base-excision-repair enzyme. Deletion of the 19-bp sequence markedly reduced the hydroxyurea (HU)-enhanced DIN7 promoter activity. In addition, nuclear fractions prepared from HU-treated cells were used in in vitro band shift assays to reveal the presence of currently unidentified transacting factor(s) that preferentially bound to the 19-bp region. These results suggest that the 19-bp sequence is a novel cis-acting element that is required for the regulation of DIN7 expression in response to HU-induced DNA damage.
Genes, 2020
Studies of mitochondrial (mt)DNA replication, which forms the basis of mitochondrial inheritance,... more Studies of mitochondrial (mt)DNA replication, which forms the basis of mitochondrial inheritance, have demonstrated that a rolling-circle replication mode exists in yeasts and human cells. In yeast, rolling-circle mtDNA replication mediated by homologous recombination is the predominant pathway for replication of wild-type mtDNA. In human cells, reactive oxygen species (ROS) induce rolling-circle replication to produce concatemers, linear tandem multimers linked by head-to-tail unit-sized mtDNA that promote restoration of homoplasmy from heteroplasmy. The event occurs ahead of mtDNA replication mechanisms observed in mammalian cells, especially under higher ROS load, as newly synthesized mtDNA is concatemeric in hydrogen peroxide-treated human cells. Rolling-circle replication holds promise for treatment of mtDNA heteroplasmy-attributed diseases, which are regarded as incurable. This review highlights the potential therapeutic value of rolling-circle mtDNA replication.
Bioscience, Biotechnology, and Biochemistry, 2008
Yeast DIN7 is a DNA damage-inducible gene. Its expression is increased in the absence of Dun1, a ... more Yeast DIN7 is a DNA damage-inducible gene. Its expression is increased in the absence of Dun1, a DNA damage checkpoint kinase. We identified a DIN7 promoter region responsible for Dun1-mediated downregulation and found that DIN7 expression was not further increased in response to hydroxyurea in Ádun1 cells. Thus DIN7 repression by Dun1 can be released upon DNA damage.
Genes to Cells, 2011
Mitochondrial fusion plays an important role in mitochondrial DNA (mtDNA) maintenance, although t... more Mitochondrial fusion plays an important role in mitochondrial DNA (mtDNA) maintenance, although the underlying mechanisms are unclear. In budding yeast, certain levels of reactive oxygen species (ROS) can promote recombination-mediated mtDNA replication, and mtDNA maintenance depends on the homologous DNA pairing protein Mhr1. Here, we show that the fusion of isolated yeast mitochondria, which can be monitored by the bimolecular fluorescence complementation-derived green fluorescent protein (GFP) fluorescence, increases the mtDNA copy number in a manner dependent on Mhr1. The fusion event, accompanied by the degradation of dissociated electron transport chain complex IV and transient reductions in the complex IV subunits by the inner membrane AAA proteases such as Yme1, increases ROS levels. Analysis of the initial stage of mitochondrial fusion in early log-phase cells produced similar results. Moreover, higher ROS levels in mitochondrial fusion-deficient mutant cells increased the amount of newly synthesized mtDNA, resulting in increases in the mtDNA copy number. In contrast, reducing ROS levels in yme1 null mutant cells significantly decreased the mtDNA copy number, leading to an increase in cells lacking mtDNA. Our results indicate that mitochondrial fusion induces mtDNA synthesis by facilitating ROS-triggered, recombination-mediated replication and thereby prevents the generation of mitochondria lacking DNA.
Genes, 2011
Mitochondria have their own genomic DNA. Unlike the nuclear genome, each cell contains hundreds t... more Mitochondria have their own genomic DNA. Unlike the nuclear genome, each cell contains hundreds to thousands of copies of mitochondrial DNA (mtDNA). The copies of mtDNA tend to have heterogeneous sequences, due to the high frequency of mutagenesis, but are quickly homogenized within a cell ("homoplasmy") during vegetative cell growth or through a few sexual generations. Heteroplasmy is strongly associated with mitochondrial diseases, diabetes and aging. Recent studies revealed that the yeast cell has the machinery to homogenize mtDNA, using a common DNA processing pathway with gene conversion; i.e., both genetic events are initiated by a double-stranded break, which is processed into 3' single-stranded tails. One of the tails is base-paired with the complementary sequence of the recipient double-stranded DNA to form a D-loop (homologous pairing), in which repair DNA synthesis is initiated to restore the sequence lost by the breakage. Gene conversion generates sequence diversity, depending on the divergence between the donor and recipient sequences, especially when it occurs among a number of copies of a DNA sequence family with some sequence variations, such as in
Molecular and Cellular Biology, 2006
Hypersuppressiveness, as observed in Saccharomyces cerevisiae, is an extremely biased inheritance... more Hypersuppressiveness, as observed in Saccharomyces cerevisiae, is an extremely biased inheritance of a small mitochondrial DNA (mtDNA) fragment that contains a replication origin (HS [rho −] mtDNA). Our previous studies showed that concatemers (linear head-to-tail multimers) are obligatory intermediates for mtDNA partitioning and are primarily formed by rolling-circle replication mediated by Mhr1, a protein required for homologous mtDNA recombination. In this study, we found that Mhr1 is required for the hypersuppressiveness of HS [ori5] [rho −] mtDNA harboring ori5, one of the replication origins of normal ([rho +]) mtDNA. In addition, we detected an Ntg1-stimulated double-strand break at the ori5 locus. Purified Ntg1, a base excision repair enzyme, introduced a double-stranded break by itself into HS [ori5] [rho −] mtDNA at ori5 isolated from yeast cells. Both hypersuppressiveness and concatemer formation of HS [ori5] [rho −] mtDNA are simultaneously suppressed by the ntg1 null mu...
Molecular biology of the cell, May 15, 2016
Mitochondria that contain a mixture of mutant and wild-type mitochondrial (mt) DNA copies are het... more Mitochondria that contain a mixture of mutant and wild-type mitochondrial (mt) DNA copies are heteroplasmic. In humans, homoplasmy is restored during early oogenesis and reprogramming of somatic cells, but the mechanism of mt-allele segregation remains unknown. In budding yeast, homoplasmy is restored by head-to-tail concatemer formation in mother cells by reactive oxygen species (ROS)-induced rolling-circle replication and selective transmission of concatemers to daughter cells, but this mechanism is not obvious in higher eukaryotes. Here, using heteroplasmic m.3243A > G primary fibroblast cells derived from MELAS patients treated with hydrogen peroxide (H2O2), we show that an optimal ROS level promotes mt-allele segregation toward wild-type and mutant mtDNA homoplasmy. Enhanced ROS level reduced the amount of intact mtDNA replication templates but increased linear tandem multimers linked by head-to-tail unit-sized mtDNA (mtDNA concatemers). ROS-triggered mt-allele segregation c...
Journal of Biological Chemistry, 2009
The abbreviations used are: dsDNA, double-stranded DNA; mtDNA, mitochondrial DNA; ssDNA, single-s... more The abbreviations used are: dsDNA, double-stranded DNA; mtDNA, mitochondrial DNA; ssDNA, single-stranded DNA; cc-dsDNA, closed circular dsDNA.
Scientific Reports
Mitochondrial (mt) DNA encodes factors essential for cellular respiration, therefore its level an... more Mitochondrial (mt) DNA encodes factors essential for cellular respiration, therefore its level and integrity are crucial. ABF2 encodes a mitochondrial DNA-binding protein and its null mutation (Δabf2) induces mtDNA instability in Saccharomyces cerevisiae. Mhr1 is a mitochondrial recombinase that mediates the predominant form of mtDNA replication and acts in mtDNA segregation and the repair of mtDNA double-stranded breaks (DSBs). However, the involvement of Mhr1 in prevention of mtDNA deletion mutagenesis is unknown. In this study we used Δabf2 mhr1-1 double-mutant cells, which lose mitochondrial function in media containing fermentable carbon sources, to investigate whether Mhr1 is a suppressor of mtDNA deletion mutagenesis. We used a suppresivity assay and Southern blot analysis to reveal that the Δabf2 mutation causes mtDNA deletions rather than an mtDNA-lacking (ρ 0) phenotype, and observed that mtDNA deletions are exacerbated by an additional mhr1-1 mutation. Loss of respiratory function due to mtDNA fragmentation occurred in ∆mhr1 and ∆abf2 mhr1-1 cells. However, exogenous introduction of Mhr1 into Δabf2 mhr1-1 cells significantly rescued respiratory growth, suggesting that Mhr1-driven homologous mtDNA recombination prevents mtDNA instability.