Molecular and Structural Basis for Replication Initiation and Strand Separation by Human Mitochondrial DNA Polymerase γ

Authors

• Viktoriia Sokolova, Thomas Jefferson UniversityFollow
• Gina Buchel, Thomas Jefferson UniversityFollow
• S. Strock, Thomas Jefferson UniversityFollow
• Ashok R. Nayak, Thomas Jefferson UniversityFollow
• Dmitry Temiakov, Thomas Jefferson UniversityFollow

Document Type

Article

Publication Date

9-22-2026

Comments

This article is the author’s final published version in Nucleic Acids Research, Volume 54, Issue 18, 2026, Article number gkag925.

The published version is available at https://doi.org/10.1093/nar/gkag925. Copyright © The Author(s) 2026.

Abstract

Defects in human mitochondrial DNA (mtDNA) replication can lead to somatic mutations associated with a range of devastating mitochondrial diseases. However, the molecular mechanisms governing the earliest steps of mtDNA replication and their fidelity remain poorly understood. Here, we found that DNA polymerase gamma (Polγ) forms stable complexes with RNA-DNA primer-template substrates, exhibiting greater stability and lower misincorporation than on DNA-primed substrates. Structural analysis revealed that Polγ interacts with the 2'-OH groups of ribose within the first four nucleotides of the primer, explaining the stability of complexes that utilize RNA primers. Although Polγ 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 Polγ is distinct from that of its yeast paralog, Mip1, and involves previously unresolved elements-the catcher and a GP loop in the exonuclease domain-that support intrinsic strand-displacement synthesis by Polγ. 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.

Recommended Citation

Sokolova, Viktoriia; Buchel, Gina; Strock, S.; Nayak, Ashok R.; and Temiakov, Dmitry, "Molecular and Structural Basis for Replication Initiation and Strand Separation by Human Mitochondrial DNA Polymerase γ" (2026). Department of Biochemistry and Molecular Biology Faculty Papers. Paper 309.
https://jdc.jefferson.edu/bmpfp/309

Creative Commons License

This work is licensed under a Creative Commons Attribution-Noncommercial 4.0 License

PubMed ID

42788360

Language

English