Ligase 3 prevents oxidative strand break-induced mitochondrial DNA loss but is not essential for replicative circularization

The mitochondrial isoform of LIG3 is proposed to catalyze both circularization of newly replicated mitochondrial DNA (mtDNA) and rejoining of free mtDNA strands in base excision and single-strand break repair. Inactivation of LIG3 has been reported to cause embryonic lethality in mice due to loss of mtDNA. Here, we applied genome editing to inactivate LIG3 in HEK 293 cells and observed only a moderate decrease of mtDNA copy numbers. BrdU incorporation experiments confirmed ongoing synthesis of intact supercoiled mtDNA. Using ultra-deep long-read sequencing of isolated mtDNA, we detected increased frequencies of single-strand and double-strand breaks clustering at sites with high GC-content, as well as hallmarks of accelerated degradation of linear mtDNA. This is likely due to the missing repair of intrinsic oxidative single-strand breaks, since the frequency of detected single-strand breaks was dependent on oxygen tension and on expression levels of enzymes involved in ROS (reactive oxygen species) defense. Exogenous oxidative challenge, that resulted in transient mtDNA damage in wild-type cells, caused dramatic mtDNA loss in LIG3-/- cell lines. Thus, our data provide evidence for the pivotal role of LIG3 in preventing mtDNA loss after oxidative damage and corroborate the hypothesis that oxidative strand break-induced mtDNA degradation is highly relevant for mtDNA turnover in vivo.