Effects of Mitochondrial Nucleases on mtDNA Degradation

Abstract

Mitochondria are unique to have a multicopy genome, resulting in a substantially different fate of damaged DNA molecules in comparison to nuclear DNA. Damaged DNA usually represents only a small fraction of total mitochondrial DNA (mtDNA) in a cell, which can be removed, through DNA degradation, without severe consequences and get replaced by replication of intact mtDNA. This idea of a "disposable genome" plays an essential role for modern gene therapy of mitochondrial diseases, which aim to eliminate pathogenic mtDNA mutations by selectively breaking down mutated mtDNA. Introducing mtDNA double-strand breaks (DSB), elimination of paternal mtDNA or virus-induced mtDNA depletion are described phenomena of eliminating mtDNA. The molecular machinery performing mtDNA degradation is still unknown. This work used the CRISPR/Cas9 technique to create stable knockout and knockin cell lines of selected mitochondrial nucleases in a cellular model to study degradation of linear mtDNA. An induced expression of restriction endonuclease mitoEagI introduced DSB into the mitochondrial genome of living cells, linearizing it in the process. Inactivation of the mitochondrial 5'–3' exonuclease MGME1 and the 3'–5' exonuclease activity of POLG (a subunit of the mitochondrial DNA polymerase gamma), through introducing the p.D274A point mutation severely impaired rapid linear mtDNA degradation. Additional knockout cell lines of other mitochondrial nucleases (APEX2, EXOG) showed no deficiencies on linear mtDNA degradation. Along with recent findings, that the mitochondrial DNA helicase Twinkle is also involved in linear mtDNA degradation (Peeva and Blei et al., 2018), this altogether proposes novel, additional roles for the mtDNA replication machinery.