Mitochondrial G4 DNA Cleavage by EndoG Activates a Flexible, Stress-Dependent Repair Response via Double strand break repair pathways

G-quadruplex (G4) structures are non-B DNA elements enriched within the mitochondrial genome and serve as substrates for Endonuclease G (EndoG). Under oxidative stress, endo G relocates from the intermembrane space to the matrix, where it cleaves G4 motifs and generates mitochondrial double-strand breaks (DSBs). Notably, mitochondrial DNA (mtDNA) frequently harbours large deletions flanked by G4 motifs; these deletions are widespread in ageing and mitochondrial disorders, yet the mechanistic basis of their formation remains poorly understood.

Here, we identify a damage-specific and stress-responsive mtDNA repair program that resolves EndoG-induced DSBs, using biochemical reconstitution and pharmacological inhibition. We show that these breaks are primarily repaired via microhomology-mediated end joining (MMEJ) and homologous recombination (HR), facilitated by the mitochondrial recruitment of canonical factors, including PARP1, MRE11, and Ligase III. Inhibition of PARP1 or MRE11 significantly impairs repair efficiency, confirming their essential roles in mitochondrial DSB resolution. Interestingly, exposure to ionising radiation (5 Gy) selectively suppresses mitochondrial MMEJ while enhancing HR, revealing a compensatory pathway switch tuned to the nature and severity of genotoxic stress. Classical nonhomologous ending (cNHEJ) remains undetectable under all conditions.

Collectively, our findings delineate a flexible, lesion-dependent mitochondrial repair network that resolves DSBs via error-prone or recombinogenic mechanisms. This work provides mechanistic insight into the origin of mtDNA deletions and highlights the adaptive plasticity of mitochondrial genome maintenance under physiological and genotoxic stress.