The role of mtDNA damage in mitochondrial dysfunction

Mammalian mitochondria have a 16.5 kb genome encoding for 13 polypeptides, 2 rRNAs, and 22 tRNAs essential for mitochondrial function. Mutations and deletions in mitochondrial DNA (mtDNA) are implicated in some hereditary diseases as well as aging, cancer and neurodegeneration. Here, we examine the role of DNA ligase in mtDNA maintenance, and the role of mtDNA damage inflicted by hydrogen peroxide (H2O2) or methyl methanesulfonate (MMS) in mitochondrial dysfunction. We establish that mitochondrial DNA ligase activity is essential for survival, persistent mtDNA damage is not sufficient to induce rapid mtDNA loss and mitochondrial dysfunction. We removed and replaced the normal mitochondrial DNA ligase III with different forms of mitochondrially-targeted DNA ligase, and found that mitochondrial DNA ligase activity is necessary for cellular survival and that any type of DNA ligase activity in the mitochondria is sufficient to maintain mtDNA integrity and copy number. To study the relationship between mtDNA integrity and mitochondrial function, we treated cells with H2O2 or with the alkylating agent MMS, both of which resulted in persistent mtDNA lesions. However, only the H2O2-treated cells showed mtDNA loss and mitochondrial dysfunction by 8 hours post-treatment, indicating that persistent mtDNA damage does not necessarily cause a rapid loss of mtDNA or mitochondrial function. These data suggest that oxidants are more efficient than alkylating agents at driving mtDNA loss and mitochondrial dysfunction.\ud We then addressed the cause of H2O2-induced loss of mtDNA 8 hours following treatment. We hypothesized that this loss of mtDNA is dependent upon mitochondrial fission and mitophagy. In order to test this hypothesis, we treated cells with the fission inhibitor mdivi-1. Mdivi-1 protected mtDNA against oxidative-induced mtDNA damage but not MMS-induced mtDNA damage. Because mdivi-1 is thought to act through inhibition of Drp1, we performed siRNA-mediated knockdown (KD) of Drp1 and observed that the knockdown did not recapitulate mdivi-1 treatment in protecting against H2O2-induced mtDNA damage. Furthermore, treating Drp1 KD cells with mdivi-1 still showed the protective effects of mdivi-1 on mtDNA damage. These results suggest that the mdivi-1 mediated protection of oxidant-induced mtDNA damage may be independent of its role in inhibiting mitochondrial fission. \u