Oxidative single-strand break formation in mitochondrial DNA

Abstract

Elevated ROS generation is associated with the pathophysiology of many neurodegenerative diseases. Since the mitochondria represent a major oxidizing environment and are considered hubs for cellular iron, this enables the Fenton-mediated reactions to generate the highly reactive hydroxyl radical (^•OH) which can cause higher levels of DNA damage in the mitochondria than in the nucleus. These DNA lesions include oxidized DNA bases, single-strand breaks (SSBs) and also rarely, double-strand breaks (DSBs). While there is some evidence for the implication of ROS-induced mtDNA damage in neurodegenerative diseases, others have argued against its contribution in somatic mtDNA mutagenesis. This controversy results from the absence of the mtDNA mutations corresponding to the most abundant modified base after oxidative stress, the oxidized guanine lesions (8-oxoG). The functional role of OGG1, considered to be the major bifunctional DNA glycosylase in removal of the 8-oxoG lesions during base excision repair (BER), remains debatable in mtDNA. Thus, we sought here, using H₂O₂, to elucidate the mechanisms of SSB formation in mtDNA by deciphering between either a physical or an enzymatic dependence for their formation during BER. To this end, I generated two OGG1 knock-outs (KOs) in HEK 293 cells using CRISPR/Cas9. Amounts of SSBs, determined by Southern blotting, a developed qPCR method as well as short-read (Illumina) and long-read (PacBio) ultra-deep sequencing to mtDNA, were comparable in both OGG1 KOs and wild-type (WT) cells upon 30 minutes of 1 mM H₂O₂ treatment. Accordingly, these findings suggested that SSBs are generated mainly by a physical attack of the ^•OH on the sugar phosphate backbone of DNA. However, the <em>in vitro</em> glycosylase (Fpg) treatment done after 30 minutes of milder 0.5 mM H₂O₂ exposure revealed a significantly higher ratio between Fpg-dependent and Fpg-independent SSBs in OGG1 KOs samples as compared to WT cells. These results indicated an inability to remove the 8-oxoG lesions in OGG1 KOs but not in WT cells after acute mild H₂O₂ exposure and proposed that a fraction of the formed SSBs upon H₂O₂ treatment enzymatically depend on the presence of OGG1. Additionally, the mtDNA degradation to DSBs, generated especially by H₂O₂ in contrast to KBrO₃, was visualized here in the D-loop region. In this regard, a possible relevant role for OGG1 was demonstrated. Collectively, results here gave an additional evidence to a functional role of OGG1 in removal of 8-oxoG in mtDNA during induced acute oxidative stress.