Fukui, H. & Moraes, C. T. Mechanisms of formation and accumulation of mitochondrial DNA deletions in aging neurons. Hum. Mol. Genet. 18, 1028-1036

Neuroscience Program, University of Miami School of Medicine, Miami, FL 33136, USA.
Human Molecular Genetics (Impact Factor: 6.39). 01/2009; 18(6):1028-36. DOI: 10.1093/hmg/ddn437
Source: PubMed


Age-dependent accumulation of partially deleted mitochondrial DNA (DeltamtDNA) has been suggested to contribute to aging and the development of age-associated diseases including Parkinson's disease. However, the molecular mechanisms underlying the generation and accumulation of DeltamtDNA have not been addressed in vivo. In this study, we have developed a mouse model expressing an inducible mitochondria-targeted restriction endonuclease (PstI). Using this system, we could trigger mtDNA double-strand breaks (DSBs) in adult neurons. We found that this transient event leads to the generation of a family of DeltamtDNA with features that closely resemble naturally-occurring mtDNA deletions. The formation of these deleted species is likely to be mediated by yet uncharacterized DNA repairing machineries that participate in homologous recombination and non-homologous end-joining. Furthermore, we obtained in vivo evidence that DeltamtDNAs with larger deletions accumulate faster than those with smaller deletions, implying a replicative advantage of smaller mtDNAs. These findings identify DSB, DNA repair systems and replicative advantage as likely mechanisms underlying the generation and age-associated accumulation of DeltamtDNA in mammalian neurons.

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    • "The integrity of mitochondrial DNA plays a critical role in maintaining cellular homeostasis and efficient repair of mtDNA damage is important for cellular survival. When DSBs are induced in mitochondria by restriction endonucleases, both intramolecular and intermolecular recombination products with large deletions are observed (Fukui and Moraes, 2009). Although homologous recombination could be one of the potential pathways of mtDNA DSB repair to ensure stability of the genome, evidence to this end in mitochondria is lacking. "
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    ABSTRACT: Mitochondrial DNA (mtDNA) deletions are associated with various mitochondrial disorders. The deletions identified in humans are flanked by short directly-repeated mitochondrial DNA sequences; however, the mechanism of such DNA rearrangements is yet to be elucidated. In contrast to nuclear DNA (nDNA), mtDNA is more exposed to oxidative damage that may result in double-strand breaks (DSBs). Although, DSB repair in nDNA is well studied, repair mechanisms in mitochondria are not characterized. In the present study, we investigate the mechanisms of DSB repair in mitochondria using in vitro and ex vivo assays. While classical-NHEJ (C-NHEJ) is undetectable, microhomology mediated alternative-NHEJ efficiently repairs DSBs in mitochondria. Interestingly, robust MMEJ (Microhomology Mediated End Joining) was observed with DNA substrates bearing 5, 8, 10, 13, 16, 19 and 22 nt microhomology. Furthermore, MMEJ efficiency was enhanced with an increase in the length of homology. Western blotting, immunoprecipitation and protein inhibition assays suggest the involvement of CtIP, FEN1, MRE11 and PARP1 in mitochondrial MMEJ. Knockdown studies, in conjunction with other experiments demonstrated that DNA LIGASE III, but not LIGASE IV or LIGASE I, is primarily responsible for the final sealing of DSBs during mitochondrial MMEJ. These observations highlight the central role of MMEJ in maintenance of the mammalian mitochondrial genome integrity and is likely relevant for deletions observed in many human mitochondrial disorders.
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    • "To study the molecular mechanisms underlying the generation and accumulation of mtDNA deletions in neurons in vivo, mice expressing neuron-specific mito-PstI were created by crossing the TRE-mito-PstI mouse with a model carrying the tTA gene under the control of the Cam- KIIa promoter (Fukui & Moraes, 2009). In the absence of Dox, CamKIIαtTA drives the expression of mito-PstI in forebrain neurons. "
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