Mitochondrial DNA damage persistence due to DNA Polymerase Gamma Disease Mutations
Authors: Krasich Rachel , Copeland William C. 
Published: November 2023

Human Mitochondrial DNA (mtDNA) is a 16.6 kb closed circular genome that is replicated by DNA polymerase γ (Polγ), the only replicative polymerase in mitochondria. Each mitochondrion contains hundreds of copies of its genome, resulting in thousands of copies per cell. Proper replication of this genome is crucial as it encodes several proteins necessary for oxidative phosphorylation, the cell's primary mode of generating ATP. Defects in replication that result in depletions, deletions, or mutations can result in decreased energy production, leading to numerous mitochondrial disorders. The gene for Polγ is one of the most commonly mutated nuclear genes in mitochondrial disease in which there are over 300 known pathogenic mutations that cause early onset mtDNA depletion syndromes and later onset diseases associated with mtDNA deletions. Environmental factors can also play a complicating role in mitochondrial diseases by exacerbating disease phenotypes. For example, it has been shown in yeast that certain Mip1 (the yeast homolog to human Polγ) mutants defective in replication have increased mutagenesis in response to MMS treatment. Our goal is to develop a human cell model to explore the effects and repair of environmentally‐induced DNA damage.MtDNA has a unique strand‐displacement mode of replication, which results in a long single‐stranded DNA (ssDNA) replication intermediate. The presence of ssDNA creates the potential for persistent lesions that can't be repaired by base‐excision repair. We have used quantitative PCR to identify any persistent lesions in mtDNA, where these lesions might occur, and if these lesions are mutagenic after MMS exposure. HEK293 cells were treated with a high dose (2 mM) of MMS for 1 hour, then allowed to recover for 5, 8, and 24 hours. Lesions were readily detectable using a quantitative PCR assay, and a low level remained persistent up to 24 hours. Deep sequencing analysis did not detect any MMS‐induced mutations, implying the lesions were either repaired or the mutation frequency was too low for detection. Cells were then treated with a range of lower doses of MMS (0.03–0.25 mM) for 8 days in an effort to increase lesion and mutation frequency. Lesions were again detectable with MMS treatment, and were equally distributed between the two halves of the genome. Deep sequencing analysis again showed no MMS‐induced mutations.Previous results in yeast implicate certain Polγ disease mutations in MMS mutagenesis. To investigate the consequences of MMS damage with human polg mutants, we exposed cells overexpressing Polγ disease alleles to MMS in the same manner as WT cells. Surprisingly, preliminary results showed Polγ mutants had no increase in MMS‐induced damage, but instead had increased lesion persistence. These results imply an important role for Polγ polymerase activity in the repair and/or tolerance of environmental damage that is separate from its exonuclease activity. Further understanding of Polγ's repair activities can potentially lead to mitigating the severity of mitochondrial disorders.