Essentiality of Mitochondrial Genome Integrity in Cell Survival

The mitochondrial genome encodes 22 tRNAs, 2 rRNAs, and 13 polypeptides that are subunits of the electron transport chain involved in oxidative phosphorylation. Because of its proximity to source of ROS generation and its non-chromatinized structure, mitochondrial genome is continuously challenged by ROS which are generated as by-products of oxidative phosphorylation, causing more damage to the mitochondrial than nuclear genome. Accumulation of somatic mitochondrial mutations has implication in the progression of aging, cancer, and neurodegenerative diseases; therefore, it is important to study the mechanisms necessary for maintaining mitochondrial integrity. ROS induce both base lesions and single-strand breaks (SSBs) containing 3’ and 5’ blocking groups in DNA. These lesions are repaired by the DNA base excision repair (BER) pathways: single nucleotide (SN) and long patch (LP)-BER. Initially, only SN-BER was thought to exist in the mitochondria, but we and others recently discovered LP-BER in the mitochondria. LP-BER differs from SN-BER in that it requires a 5’ end cleaning enzyme to remove the 5’ blocking group which hinders DNA polymerase activity after removal of damaged base and 3’ blocking group by DNA glycosylase and APE1, respectively. Currently, three 5’ end cleaning enzymes in the mitochondria have been proposed: EXOG, DNA2, and FEN1. EXOG is the only endo/exonuclease that exists exclusively in the mitochondria, and my results suggest that EXOG is the predominant 5’ end cleaning enzyme in mitochondrial LP-BER. Immunoprecipitation of EXOG-FLAG immunocomplex showed the presence of other mitochondrial LP-BER proteins. Unlike FEN1 and DNA2, EXOG downregulation in HeLa and HCT116 p53 +/+ cells by siRNA transfection caused significant accumulation of SSBs/repair intermediates in the mitochondrial genome, depolarization of mitochondrial membrane potential, increase in ROS generation, and cell death. Increase in Annexin V positive cell population and cleavage of caspase-3 and PARP suggest cell death via apoptosis. In addition, EXOG downregulation in A549 control and A549 rho0 cells, which are depleted of mitochondrial DNA, induced cell death in A549 control but not in A549 rho0 cells, suggesting that damage and downstream breakage of mitochondrial DNA are necessary for activating cell death. Therefore, EXOG is crucial in maintaining mitochondrial genome integrity, which is essential in cell survival. Furthermore, decrease in mitochondrial BER activity and increase in oxidative stress with age in mouse skeletal muscles compared to other tissues further demonstrate that maintenance of mitochondrial DNA repair may be important in slowing down the progression of sarcopenia.