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However, the molecular mechanisms governing the earliest steps of mtDNA replication and their fidelity remain poorly understood. Here, we found that DNA polymerase gamma (Pol\u03b3) forms stable complexes with RNA\u2013DNA primer\u2013template substrates, exhibiting greater stability and lower misincorporation than on DNA-primed substrates. Structural analysis revealed that Pol\u03b3 interacts with the 2\u2032-OH groups of ribose within the first four nucleotides of the primer, explaining the stability of complexes that utilize RNA primers. Although Pol\u03b3 requires TWINKLE to extend RNA primers, its intrinsic strand-displacement activity allows it to extend DNA primers independently. Structural data further show that the strand-separation mechanism in human Pol\u03b3 is distinct from that of its yeast paralog, Mip1, and involves previously unresolved elements\u2014the catcher and a GP loop in the exonuclease domain\u2014that support intrinsic strand-displacement synthesis by Pol\u03b3. Structure-guided mutagenesis of elements involved in strand separation supports these structural observations. Together, our study provides mechanistic insight into mtDNA replication initiation and strand separation and has implications for understanding the molecular basis of mitochondrial disease.<\/jats:p>","DOI":"10.1093\/nar\/gkag925","type":"journal-article","created":{"date-parts":[[2026,9,15]],"date-time":"2026-09-15T12:00:54Z","timestamp":1789473654000},"source":"Crossref","is-referenced-by-count":0,"title":["Molecular and structural basis for replication initiation and strand separation by human mitochondrial DNA polymerase \u03b3"],"prefix":"10.1093","volume":"54","author":[{"given":"Viktoriia","family":"Sokolova","sequence":"first","affiliation":[{"name":"Department of Biochemistry and Molecular Biology, Thomas Jefferson University ; 1020 Locust St, Philadelphia, PA 19107 ,","place":["United 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