Accumulation of ΔmtDNA has been observed in (i) mitochondrial diseases, which are caused by non-Mendelian inheritance of maternal mutated mtDNA or inheritance of mutant nuclear genes associated with the maintenance of mtDNA and (ii) mammalian aging. In both scenarios, the characteristics of ΔmtDNAs are similar (2
). Deletions often involve the major arc of mtDNA, between the major origins of replication. They are either flanked by direct repeats (one of which is removed by the deletion process) or no direct repeats. Therefore, similar molecular mechanisms may underlie the generation of deletions in these scenarios.
As discussed in a recent review, replication errors and repair of damaged mtDNA are the two potential mechanisms that may involve in the formation of ΔmtDNA (10
). While recombination of mtDNA during replication still stands as a valid model, this model could only account for generation of deletions with direct repeats or homology-dependent recombination.
Krishnan et al
) proposed that 3′–5′ exonuclease activity following the introduction of DSB within the major arc exposes single-stranded regions of mtDNA, which subsequently undergoes homologous annealing at micro-domains containing direct repeats. Although this model remains attractive, it only accounts for homology-dependent recombination. In addition, previous work from our laboratory on muscle-specific constitutive mitoPstI transgenic mice showed that DSB-induced deletions contained short or no direct repeats (12
). This work proposed that single-stranded 3′ free ends generated by DSB could anneal with the end of the unpaired D-loop strand and initiate recombination, through short homologous sequences.
One shortcoming of the previous model, however, was the constitutive activity of mitoPstI throughout the life of mice and the resulting severe depletion of mtDNA. This scenario compromised cell viability and was unlikely to be suitable for the accumulation of ΔmtDNA. In the present study, we generated an improved mouse model, in which the expression of mitoPstI and therefore the induction of DSB can be spatially and temporarily regulated. Three-day transient removal of Dox from young adult mitoPstI/tTA double transgenic mice successfully induced the formation of ΔmtDNA without a noticeable depletion of mtDNA. Because our identification of ΔmtDNA relied on the cloning of PCR products, it is likely that our technique identified only a subset of total ΔmtDNA species. Nevertheless, our detection and characterization of ΔmtDNA in neurons revealed the formation of a variety of deletions with various sizes (6.3–12.5 kb) with and without direct repeats, a pattern also found in naturally occurring age-related ΔmtDNA. These results suggest that DSBs in neuronal mtDNA lead to both homology-dependent and independent recombination. DSB in mtDNA can occur by several processes, including replication stalling (19
). The 3′ and 5′ends of breakpoints were proximal to the PstI-recognition sites in some deletions, but were quite distal in others. The formation of deletions such as Deletions 20–22, which involved recombination of 3′ end proximal to the Pst
I site and the D-loop region, could be explained by a preferential recombination of free ends at the D-loop (12
). However, the formation of other deletions is difficult to explain without the participation of exonuclease activity (10
). We propose that NHEJ also plays a role in homology-independent recombination. Although NHEJ machineries have not been identified in neuronal mitochondria (23
), Deletion 3 formed by blunted ends of two Pst
I-recognition sites is likely to be mediated by this mechanism. Exonucleases such as POLG (10
) may degrade single-strand ends generated by DSB, and intermediates may undergo NHEJ in a stochastic manner, forming ΔmtDNA without the involvement of direct repeats. POLG is an mtDNA polymerase with proofreading 3′–5′ exonuclease activity and may be involved in this process. However, knockin mice carrying homozygous mutant POLG with proofreading defects also accumulate large-scale deletions with no direct repeats (24
). Furthermore, it has not been confirmed that POLG has a capacity to degrade a large portion of mtDNA. Therefore, it is possible that other uncharacterized exonuleases participate in the formation of deletions. Identification and characterization of those exonucleases may provide an insight in preventing the formation and accumulation of ΔmtDNA molecules during aging.
In aged muscle, it has been shown that accumulation of ΔmtDNA occurs through clonal expansion of limited numbers of ΔmtDNA formed stochastically along the course of aging (10
). However, the molecular mechanisms that account for this clonal expansion has been poorly understood. The proposed mechanisms include (i) random genetic drift (25
) and (ii) replicative advantage of ΔmtDNA (26
). Limitations of the former mechanism were recently discussed by Krishnan et al
). Although we have described ex vivo
data suggesting a replicative advantage of ΔmtDNA (26
), there has been no experimental in vivo
evidence supporting the replicative advantage of ΔmtDNA molecules in post-mitotic cells. Our data, albeit limited to a few mice, suggest that after being formed by DSB, mtDNAs with large deletions accumulate preferentially when compared with mtDNAs with smaller deletions. This observation demonstrates that the accumulation of ΔmtDNA during neuronal aging is not merely due to mtDNAs defective properties, implying a replicative advantage of smaller mtDNAs.