Ultra-Deep Sequencing of Mouse Mitochondrial DNA: Mutational Patterns and Their Origins

Abstract

Somatic mutations of mtDNA are implicated in the aging process, but there is no universally accepted method for their accurate quantification. We have used ultra-deep sequencing to study genome-wide mtDNA mutation load in the liver of normally- and prematurely-aging mice. Mice that are homozygous for an allele expressing a proof-reading–deficient mtDNA polymerase (mtDNA mutator mice) have 10-times-higher point mutation loads than their wildtype siblings. In addition, the mtDNA mutator mice have increased levels of a truncated linear mtDNA molecule, resulting in decreased sequence coverage in the deleted region. In contrast, circular mtDNA molecules with large deletions occur at extremely low frequencies in mtDNA mutator mice and can therefore not drive the premature aging phenotype. Sequence analysis shows that the main proportion of the mutation load in heterozygous mtDNA mutator mice and their wildtype siblings is inherited from their heterozygous mothers consistent with germline transmission. We found no increase in levels of point mutations or deletions in wildtype C57Bl/6N mice with increasing age, thus questioning the causative role of these changes in aging. In addition, there was no increased frequency of transversion mutations with time in any of the studied genotypes, arguing against oxidative damage as a major cause of mtDNA mutations. Our results from studies of mice thus indicate that most somatic mtDNA mutations occur as replication errors during development and do not result from damage accumulation in adult life.

The role of mitochondria in cardiac development and protection

“…In addition, 8-oxo-dG's are read as dA's during replication and will result in G>A mutations as observed in the mutation pattern of Sod2 +/-mouse hearts [28]. Interestingly, this is not the case in ageing wild-type mice, which seem to accumulate mtDNA mutations caused by replication errors, thus speaking for an efficient repair of oxidative damage in most tissues [98]. Although high levels of mtDNA mutations do clearly impair OXPHOS, they do not increase ROS production [99], undermining the old hypothesis of a mitochondrial vicious circle of ROS and mtDNA damage [100].…”

Organelle DNA rearrangement mapping reveals U-turn-like inversions as a major source of genomic instability in Arabidopsis and humans

“…For example, it was shown that the mutation of the type I polymerase POLIB in Arabidopsis causes replication stress at early developmental stages and increases the amount of DSBs upon genotoxic stress treatment (Parent et al 2011). Interestingly, mutation of the mammalian mitochondrial DNA polymerase gamma has also been linked to replication stress and DSBs (Vermulst et al 2008;Ameur et al 2011). Replication-dependent DSBs are known to arise when a fork collapses or encounters a nick in the matrix DNA (Zeman and Cimprich 2014).…”

Visualization of Mitochondrial Respiratory Function using Cytochrome <em>C</em> Oxidase / Succinate Dehydrogenase (COX/SDH) Double-labeling Histochemistry

“…The mitochondrial theory of aging suggests a role for mtDNA mutations, which can alter bioenergetics homeostasis and cellular function, in the aging process 3 . A wealth of evidence has been compiled in support of this theory 1,4 , an example being the mtDNA mutator mouse 5 ; however, the precise role of mtDNA damage in aging is not entirely understood 6,7 .Observing the activity of respiratory enzymes is a straightforward approach for investigating mitochondrial dysfunction. Complex IV, or cytochrome c oxidase (COX), is essential for mitochondrial function.…”