Mitochondria in eukaryotic cells play a primary role in the production of ATP—the cellular source of energy—through the oxidative phosphorylation system. Mitochondria have their own genome (mtDNA), whose copy number varies in response to the physiological environment surrounding the cell (1
). Genes in mtDNA, as well as in nuclear genomes, encode essential protein subunits that participate in oxidative phosphorylation. mtDNA is constantly subjected to oxidative damage due to reactive oxygen species (ROS), which are byproducts of oxidative phosphorylation. This damage results in mutations in the mtDNA, which are associated with aging and various mitochondrial disorders (2
). On the other hand, recent evidence suggests that ROS regulate mtDNA replication: e.g. the treatment of human cells with hydrogen peroxide results in a transient increase in mtDNA copy number (3
), and the amount of mtDNA correlates with the ROS levels in cell lines carrying different mouse mtDNA haplotypes (4
). Because decreased cellular levels of mtDNA clinically correlate with mitochondrial disorders, such as mtDNA depletion syndrome, investigators are seeking to elucidate the mechanisms that regulate mtDNA copy number (5
). Studies on yeast and human cells have shown that mtDNA-related nuclear gene expression and the cellular levels of deoxyribonucleoside triphosphates (dNTPs) play critical roles in the regulation of mtDNA copy number (6
). Whether mitochondria contain mechanisms that regulate mtDNA copy number in response to ROS, however, is currently unknown (7
In the yeast Saccharomyces cerevisiae
the replication and segregation of mtDNA depend on the homologous DNA pairing protein Mhr1, which is required for mtDNA recombination (8
). Generally, DNA recombination is initiated at a double-stranded break (DSB); after the break occurs, a homologous DNA pairing protein directs the formation of a heteroduplex joint (‘the DSB repair model’ of recombination) (9
). Our studies revealed that the excision-repair enzyme Ntg1 creates a DSB at ori5
, one of the active replication origins in yeast mtDNA; the DSB level increased when yeast cells were exposed to oxidative stress (10
). These studies suggested that by recognizing a specific oxidative modification at ori5
, Ntg1 induces DSB to initiate rolling-circle mtDNA replication, as well as recombination, through the homologous DNA pairing activity of Mhr1 (10
). Ntg1 initiates base-excision repair of oxidative DNA damage by excising damaged bases via its DNA-N-glycosylase activity; subsequently, via its DNA lyase activity, it introduces single-stranded breaks (nicks) in the double-stranded DNA (11
). Based on our previous studies showing a role for Ntg1 in the initiation of mtDNA replication, as well as a correlation between the ROS levels in mitochondria and the mtDNA copy number, we sought to uncover the molecular mechanisms underlying ROS-controlled changes in mtDNA copy number.
The Mec1/Rad53 nuclear checkpoint pathway, which is activated by nuclear DNA damage, increases mtDNA copy number by affecting the activity of ribonucleotide reductase and thereby the cellular pool of dNTPs (12
). ROS are generated from either the electron transport chain or the metabolic enzyme α-ketoglutarate dehydrogenase in the tricarboxylic acid cycle; both of these ROS sources are found in normally functioning mitochondria (13
). In this study, to eliminate the effects of both the Mec1/Rad53 nuclear checkpoint pathway and possible regulatory control by nuclear genes on the amounts of ROS and mtDNA, we employed isolated mitochondria harboring hypersuppressive (HS) [ori5
] mtDNA, treated with various concentrations of hydrogen peroxide, as a model system to investigate the direct effects of ROS on mtDNA copy number. Hydrogen peroxide has been used as a ROS-generating agent in a variety of studies about the effects of ROS on cellular functions.
In this study, we show that exposing isolated yeast mitochondria to hydrogen peroxide results in simultaneous increases in Ntg1-dependent DSB levels at ori5 and in mtDNA copy number, an effect that is dependent on Ntg1 and Mhr1. It is likely that ROS trigger Ntg1- and Mhr1-dependent mtDNA replication, which leads to an increase in mtDNA copy number.