Study of mitochondrial mutagenesis induced by hydrogen peroxide and methyl-methanesulfonate in mouse fibroblasts

Abstract

Mitochondria is an organelle present in most eukaryotes that plays a key role in energy generation processes, being essential for cellular homeostasis. Mitochondrial DNA (mtDNA) encodes 37 genes, including 2 ribosomal RNAs, transport RNAs and 13 polypeptides, components of 4 of the five oxidative phosphorylation protein complexes. Somatic mutations in this genome accumulate in cancer and normal aging in mammals, which shows the importance of maintaining its integrity. In studies with different organisms, it was found that mtDNA has higher mutation rates than nuclear DNA, but the causes and mutagenic pattern of this phenomenon have not been completely elucidated. The mitochondrial theory of aging proposes that the proximity between mtDNA and the electron transport chain leaves the mitochondrial genome more exposed to oxidizing species, which accumulate mutations over time, resulting in dysfunctional organelles. In this model, oxidants generated during electron transport are considered the main endogenous mutagenic agents of mtDNA. However, several recent results have not found the predicted mutagenic pattern for oxidative damage and do not support this theory. On the other hand, studies with transgenic mice expressing DNA polymerase gamma (Pol ³) with deficient exonuclease activity (named mutator mice) demonstrated that replicative errors can generate an expressive accumulation of point mutations and deletions in mtDNA, resulting in an accelerated aging phenotype and decreased lifespan. The importance of Pol ³ in mutations originated in the replicative process has been demonstrated, but its role in lesion-induced mutagenesis has not yet been investigated. In addition, the presence of DNA polymerase beta (Pol ²) in mitochondria was recently demonstrated and it's very likely to be involved in mtDNA base excision repair. Therefore, we propose to investigate lesion-induced mutagenesis in mtDNA and the role of Pol ³ and Pol ² in this process. The study will be performed with primary fibroblasts from wild-type C57/B16 mice; wild-type with decreased expression of Pol ² and Pol ³ exonuclease deficient (mutator mice derivatives). Cells will be exposed to sub-lethal doses of two genotoxins: hydrogen peroxide and methyl-methanesulfonate, which generate oxidized and alkylated bases, respectively. For each treatment, the following will be measured: i) lesion burden and repair rates using Long-range qPCR; ii) mutation rate using Random Mutation Capture Assay; iii) the types of mutation by automated Sanger sequencing.