The mutational spectrum of the mitochondrial DNA (mtDNA) does not resemble any of the known mutational signatures of the nuclear genome and variation in mtDNA mutational spectra between different organisms is still incomprehensible. Since mitochondria are responsible for aerobic respiration, it is expected that mtDNA mutational spectrum is affected by oxidative damage. Assuming that oxidative damage increases with age, we analyse mtDNA mutagenesis of different species in regards to their generation length. Analysing, (i) dozens of thousands of somatic mtDNA mutations in samples of different ages (ii) 70053 polymorphic synonymous mtDNA substitutions reconstructed in 424 mammalian species with different generation lengths and (iii) synonymous nucleotide content of 650 complete mitochondrial genomes of mammalian species we observed that the frequency of AH > GH substitutions (H: heavy strand notation) is twice bigger in species with high versus low generation length making their mtDNA more AH poor and GH rich. Considering that AH > GH substitutions are also sensitive to the time spent single-stranded (TSSS) during asynchronous mtDNA replication we demonstrated that AH > GH substitution rate is a function of both species-specific generation length and position-specific TSSS. We propose that AH > GH is a mitochondria-specific signature of oxidative damage associated with both aging and TSSS.

It has been shown recently that mitochondrial (mtDNA) somatic variants are numerous enough to trace cellular lineages in our body. Here we extend this statement and demonstrate that mtDNA variants can be interpreted not only as neutral markers of cell divisions but the relative frequency of different mtDNA substitutions (i.e. mtDNA mutational spectrum) can inform us about important biological properties such as cell longevity. Analysing 7611 somatic mtDNA mutations from 37 types of human cancers and more than 2000 somatic mtDNA mutations from 25 healthy human tissues we observed that mtDNA mutational spectrum is associated with cell turnover rate: the ratio of T>C to G>A is increasing with cell longevity. To extend this logic we considered that, if universal, the discovered mutation bias may drive the differences in mtDNA mutational spectrum between mammalian species with short-(‘mice’) and long-(‘elephants’) lived oocytes. Based on presumably neutral polymorphisms in MT-CYB we reconstructed mutational spectra for 424 mammalian species and obtained that the fraction of T>C positively correlated with the species-specific generation length, which is a good proxy for oocyte longevity. Next, comparing complete mitochondrial genomes of 650 mammalian species we confirmed that exactly the same process shapes the nucleotide content of the most neutral sites in the whole mitochondrial genomes of short-(high T, low C) versus long-(low T, high C) lived mammals. Altogether analysing mtDNA mutations in time interval from dozens of years (somatic mutations) through the hundreds of thousands of years (within species polymorphisms) to millions of years (between species substitutions) we demonstrated that T>C/G>A positively correlates with cellular and organismal longevity. We hypothesize that the discovered mtDNA signature presents a chemical damage which is associated with the level of oxidative metabolism which, in turn, correlates with cellular and organismal longevity. The described properties of mtDNA mutational spectrum shed light on mtDNA replication, mtDNA evolution of mammals and can be used as a marker of cell longevity in single-cell analyses of heterogeneous samples.

Mitochondrial mutational signature is very conserved and low deviations between species have been associated with longevity. By reconstructing species-specific mtDNA mutational spectrum for ray-finned fishes (Actinopterygii), we observed that temperature is a strong additional factor shaping the mtDNA mutational spectrum in ectotherms. The analysis of mammalian endotherms, with a special focus on species with temporarily or permanently low metabolic rates (hibernators, daily torpors, naked mole rat, etc.), confirmed the temperature effect, suggesting that two main factors shape between-species variation in mitochondrial mutational spectra: longevity and temperature.

Background Aging in postmitotic tissues is associated with clonal expansion of somatic mitochondrial deletions, the origin of which is not well understood. Such deletions are often flanked by direct nucleotide repeats, but this alone does not fully explain their distribution. Here, we hypothesized that the close proximity of direct repeats on single-stranded mitochondrial DNA (mtDNA) might play a role in the formation of deletions. Results By analyzing human mtDNA deletions in the major arc of mtDNA, which is single-stranded during replication and is characterized by a high number of deletions, we found a non-uniform distribution with a “hot spot” where one deletion breakpoint occurred within the region of 6–9 kb and another within 13–16 kb of the mtDNA. This distribution was not explained by the presence of direct repeats, suggesting that other factors, such as the spatial proximity of these two regions, can be the cause. In silico analyses revealed that the single-stranded major arc may be organized as a large-scale hairpin-like loop with a center close to 11 kb and contacting regions between 6–9 kb and 13–16 kb, which would explain the high deletion activity in this contact zone. The direct repeats located within the contact zone, such as the well-known common repeat with a first arm at 8470–8482 bp (base pair) and a second arm at 13,447–13,459 bp, are three times more likely to cause deletions compared to direct repeats located outside of the contact zone. A comparison of age- and disease-associated deletions demonstrated that the contact zone plays a crucial role in explaining the age-associated deletions, emphasizing its importance in the rate of healthy aging. Conclusions Overall, we provide topological insights into the mechanism of age-associated deletion formation in human mtDNA, which could be used to predict somatic deletion burden and maximum lifespan in different human haplogroups and mammalian species.