Persistence of large mtDNA rearrangements linked to premature aging in Pol γ exonuclease-deficient mice

Mitochondrial DNA mutations are hallmarks of aging. MtDNA in all opisthokonts is replicated exclusively by DNA Polymerase γ (Pol γ; encoded by POLG). PolgD257A/D257A mice, lacking Pol γ exonuclease proofreading (exo⁻), exhibit premature aging and higher mtDNA mutation rates than Polgwt/wt (exo⁺) mice. Using short-read sequencing and the ultra-sensitive LostArc indel-junction detection pipeline, we analyzed mtDNA from exo⁻ and exo⁺ mice across ten tissues. Indel-junction frequency, endpoint sequence context, and inferred indel size varied systematically by tissue and exonuclease state. Exo⁻ tissues, especially post mitotic tissues, evidenced exceptionally high burdens of mtDNA Large Indels (mtDNALIs). These create covalently closed, circular, double-stranded mtDNA that persists despite often lacking replication origins. This suggests limited removal of aberrant circular dsDNA, particularly in post-mitotic tissues. MtDNALI endpoint sequence contexts in exo⁺ and exo⁻ tissues mimic those in young and elderly human muscle and are dominated by sequence-dependent and -independent indel mechanisms, respectively. Long-read sequencing recapitulated short-read junction patterns, including endpoint concentration near the 7S DNA 3’ end, but also revealed mtDNA circles with multiple mtDNALIs, including deletions and duplications. Together, these results implicate accumulation of mtDNALIs, compounded by insufficient mechanisms for eliminating closed circular aberrant mtDNA, as contributing to the premature aging phenotype.