Our analysis has revealed a number of novel aspects of the accumulation of mtDNA mutations in wildtype and mtDNA mutator mice. The mutators show highly elevated point mutation frequencies as compared to their wtmut
siblings, consistent with previous results 
. The SOLiD sequencing estimates of mutation loads presented here are similar to the mutation load estimates that we and others previously have obtained by Sanger sequencing of cloned PCR fragments 
. In a recent study using a different next-generation DNA sequencing technology, He et al. 
reported eight sites with a mutation frequency >1.6% in a human mtDNA sample. If we apply their detection threshold to our data, we identify five such sites in the wtB6
mice. Techniques that enable identification of low frequency variants, such as those used in our study, are likely to uncover additional variability and provide a more complete understanding of the mitochondrial mutation load.
We hypothesized that our analysis criteria should also uncover gradual increase in the mutation loads with natural aging, consistent with published results obtained by other mutation detection methods (reviewed in 
). However, we neither detected a difference in the mtDNA mutation load in liver mtDNA from wtB6
mice at different ages (30–84 weeks), nor did we see a shift in the mutational spectrum consistent with oxidative damage causing mtDNA mutations in mice of the different genotypes. Together, the data we present here suggest that most mtDNA mutations are due to mtDNA replication errors and that oxidative damage of mtDNA does not drive the aging process in liver.
Our estimates of the mutation load in the wtB6
mice are in good agreement with estimates based on sequencing of cloned PCR fragments 
, but they are higher than estimates obtained by a restriction enzyme digestion-based assay 
(summarized in Table S6
). Also, we observed no increase in mutation load in wtB6
mice with age, as reported elsewhere 
. A possible explanation for this difference from results in the literature is that the next-generation sequencing method has an inherent limitation in detecting extremely low levels of mutations. It remains possible that there is a slight increase of mutation load with age in wtB6
mice, as reported in other studies 
, and the true mutation levels may be below the detection threshold of the SOLiD sequencing method. But in any case, the mutation levels seen in the aging wtB6
mice were very modest in comparison to those of age-matched mtDNA mutator mice. Also, we chose to only analyze mtDNA from mouse liver as this tissue made it possible for us to obtain sufficient quantities of pure mtDNA for direct sequencing, without excess DNA amplification. A continuously dividing tissue like liver may show a different pattern of accumulation of mtDNA point mutations with age in comparison with a postmitotic tissue such as brain or heart.
The different coding regions showed similar mutation frequencies, while the control region had a much lower mutation frequency. A reduction in the overall mutation load has previously been observed in the mitochondrial control region of mtDNA mutator mice 
. The control region contains crucial sequence elements controlling replication and transcription of mtDNA 
. It is therefore likely that mutations that inhibit mtDNA maintenance or expression could undergo strong selection and be eliminated from the mtDNA pool.
Some of the mtDNA point mutations observed in siblings to mtDNA mutator mice are likely to have been inherited via the maternal gamete of their heterozygous mother. The wtmut
mice from this cross carry approximately twice the number of high frequency point mutations (>0.5%) in comparison with wtB6
mice. In addition, an elevated number of small indel mutations are observed in the wtmut
mice, but not in wtB6
mice (Table S4
). We cannot exclude that some of the shared point mutations represent extreme mutational hotspots, however, a more likely explanation is that these shared mutations are maternally inherited. A similar phenomenon has been observed in humans, where the variability at several positions was shown to be inherited from the common maternal cytoplasm instead of representing repeated de novo
mutational events 
In mtDNA mutator mice, approximately 30% of the mtDNA molecules are non-replicating, linear mtDNA molecules with large deletions 
. It could be speculated that these molecules contain an elevated mutation load and that most of the mutation load is sequestered in these molecules. By ultra-deep sequencing, we were able to determine that the mutation load in the region covering the linear molecule did not vary from the global mtDNA mutation load in the mutators. A recent publication suggests the PolgAmut
polymerase may pause at the control and OriL regions during mtDNA replication, which may explain the generation of the linear molecules with large deletions 
. Our results are consistent with a hypothesis that altered processivity of the mutator polymerase and not the point mutations per se
, are responsible for the creation of the linear molecules. The physiological consequences of the linear molecules, and their contribution to the premature aging in the mtDNA mutator mice, remain unclear and worthy of further investigation.
Circular mtDNA molecules with deletions have been suggested to be the driving force of the aging phenotype in the mutator mice, and are reliably detected in human tissues during aging. High levels of these circular mtDNA molecules with deletions lead to mitochondrial dysfunction in human patients 
or mice engineered to contain these mutations 
. However, we found that the circular mtDNA molecules with deletions are exceedingly rare in mtDNA mutator mice, with only 4 breakpoints being detected in the millions of reads of two mutator samples. Very low levels of this type of deleted molecules have also been reported by studies using different PCR based analyses 
. Recently, an independent next-generation sequencing analysis detected exceedingly low levels of this type of mutation in brain and heart of mtDNA mutator mice 
. Large deletion of mtDNA are known to impair mitochondrial translation due to lack of one or more tRNA genes 
. However, mtDNA mutator mice do not display impaired mitochondrial translation in heart or liver 
and the levels of deleted mtDNA are much lower than the levels observed in respiratory chain deficient mouse strains with single 
and multiple 
deletions of mtDNA. Together, these observations provide strong evidence that the circular deleted mtDNA molecules are not the causative factor in the aging phenotype of the mtDNA mutator mice. A recent study made a remarkable observation that similar low levels of circular deletions were accumulating in a mouse model with a tissue-specific disruption of the mitochondrial fusion process 
. The presence of very low levels of circular mtDNA molecules with deletions in two very different models of mitochondrial dysfunction suggests these rare events are being generated as a secondary consequence of mitochondrial dysfunction. Another possibility is that these molecules are continuously generated at low frequency during normal mtDNA replication and that mitochondrial dysfunction limit their clearance.
The large numbers of point mutations in adult mtDNA mutator mice result in production of highly mutated mtDNA-encoded respiratory chain subunits, causing the experimentally observed instability of the respiratory chain complexes 
. There is likely a threshold for the tolerance of point mutations, where eventually the combined effect of the many amino acid changes in mtDNA mutator mice cause destabilization of respiratory chain complexes and lead to mitochondrial dysfunction. Our results support the assertion that the accumulation of point mutations has an adversary effect on mitochondrial function and cause the premature aging syndrome in the mtDNA mutator mice.