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Mitochondrial DNA (mtDNA) mutations are thought to have a causal role in many age-related pathologies. Here we identify mtDNA deletions as a driving force behind the premature aging phenotype of mitochondrial mutator mice, and provide evidence for a homology-directed DNA repair mechanism in mitochondria that is directly linked to the formation of mtDNA deletions. In addition, our results demonstrate that the rate at which mtDNA mutations reach phenotypic expression differs markedly among tissues, which may be an important factor in determining the tolerance of a tissue to random mitochondrial mutagenesis.

Whether mitochondrial mutations cause mammalian aging, or are merely correlated with it, is an area of intense debate 1 . Here, we use a new, highly sensitive assay 2 to redefine the relationship between mitochondrial mutations and age. We measured the in vivo rate of change of the mitochondrial genome at a single‐base pair level in mice, and we demonstrate that the mutation frequency in mouse mitochondria is more than ten times lower than previously reported. Although we observed an 11-fold increase in mitochondrial point mutations with age, we report that a mitochondrial mutator mouse 3 was able to sustain a 500-fold higher mutation burden than normal mice, without any obvious features of rapidly accelerated aging. Thus, our results strongly indicate that mitochondrial mutations do not limit the lifespan of wild-type mice. Mitochondria are functionally diverse organelles with a central role in both oxidative phosphorylation 4 and apoptosis 5 . Accordingly, postmitotic tissues with high rates of oxygen consumption, such as brain and heart, are exceptionally sensitive to mitochondrial dysfunction 6 . The mitochondrial theory of aging postulates that a lifelong accumulation of mitochondrial DNA (mtDNA) mutations in multiple tissues eventually results in mitochondrial failure, which, in synergy with downstream processes such as apoptosis, results in loss of cellularity and the progressive decline of tissue functioning known as aging 3,7 . These mutations may compromise the integrity of the electron transport chain and increase the formation of reactive oxygen species (ROS), creating a vicious cycle of mutagenesis that continuously amplifies the production of cytotoxic oxygen radicals 8,9 . Although several studies have quantified either tissue-specific mutation frequencies 10 or mutation accumulation as a function of age 11 , most methods rely heavily on PCR and cloning-based strategies 12,13 , techniques that are limited in throughput and that may be confounded by polymerase infidelity on damaged templates and by cloning artifacts. Therefore, we explored the relationship between age and mutation accumulation in mtDNA with an adaptation of the random mutation capture (RMC) assay 2,14 , a quantitative