{
  "abstract": "Mitochondrial DNA (mtDNA) maintenance is essential for cellular homeostasis, and defects in mtDNA replication are linked to a broad spectrum of mitochondrial diseases. During replication, DNA polymerase {gamma} (Pol{gamma}) must traverse duplex junctions and stable secondary structures, yet how the human enzyme overcomes these barriers remains incompletely understood. Here, cryo-electron microscopy structures of Pol{gamma} bound to forked DNA, G-quadruplex (G4)-containing DNA and DNA bound to mitochondrial single-stranded DNA-binding protein (mtSSB) reveal a common template-entry path along the catcher domain across distinct substrate and active-site configurations. Within this domain, an arginine-rich helix containing R1026, R1030 and R1034 constitutes a Template Stabilising Motif (TSM). Biochemical reconstitution and DNA-binding experiments show that disrupting the TSM selectively impairs strand displacement, RNA-DNA hybrid displacement and synthesis through G4-forming sequences, while largely preserving synthesis on unstructured templates. Single-molecule optical-tweezers experiments further show that mechanical destabilisation of the fork partially restores mutant strand-displacement activity, whereas force or mtSSB restores primer-extension kinetics on ssDNA templates. Together, these findings establish the role of the TSM in maintaining productive template engagement and identify template stabilisation as a common mechanism enabling Pol{gamma} to traverse structurally diverse barriers in mtDNA.",
  "abstract_shown": true,
  "affiliations": [
    "Spanish National Cancer Research Centre (CNIO), Melchor Fernandez Almagro 3, 28029, Madrid, Spain"
  ],
  "article_type": "New results",
  "arxiv": null,
  "author_total": 10,
  "authors": [
    {
      "name": "S. Miguez-Amil"
    },
    {
      "name": "A. Grande-Garcia"
    },
    {
      "name": "I. Plaza Garcia Abadillo"
    },
    {
      "name": "M. Moreno-Morcillo"
    },
    {
      "name": "E. Casajus-Pelegay"
    },
    {
      "name": "E. Arean-Ulloa"
    },
    {
      "name": "C. Chacon-Sanchez"
    },
    {
      "name": "G. L. Ciesielski"
    },
    {
      "name": "B. Ibarra"
    },
    {
      "affs": [
        0
      ],
      "name": "R. Fernandez-Leiro"
    }
  ],
  "bibtex": "https://research.draconitic.com/paper/d3j6nn4g5zy1m.bib",
  "cited_by": 0,
  "doi": "10.64898/2026.09.27.754746",
  "fulltext_url": "https://www.biorxiv.org/content/10.64898/2026.09.27.754746v1.full",
  "id": "d3j6nn4g5zy1m",
  "issns": [],
  "landing_url": "https://www.biorxiv.org/content/10.64898/2026.09.27.754746v1",
  "language": null,
  "licence": "CC-BY-NC-ND",
  "licence_label": "CC BY-NC-ND",
  "licence_source": "article",
  "licence_url": "https://creativecommons.org/licenses/by-nc-nd/4.0/",
  "metadata_licence": null,
  "native_subjects": [
    [
      "biorxiv",
      "molecular biology"
    ],
    [
      "openalex",
      "1312|DNA and Nucleic Acid Chemistry"
    ]
  ],
  "openalex": "W7214630793",
  "pdf_url": "https://www.biorxiv.org/content/10.64898/2026.09.27.754746v1.full.pdf",
  "pmcid": null,
  "pmid": null,
  "published": "2026-09-27",
  "publisher": null,
  "ris": "https://research.draconitic.com/paper/d3j6nn4g5zy1m.ris",
  "sources": [
    {
      "first_seen": "2026-10-30T12:24:52Z",
      "native_id": "10.64898/2026.09.27.754746",
      "source": "biorxiv",
      "url": "https://www.biorxiv.org/content/10.64898/2026.09.27.754746v2"
    }
  ],
  "subjects": [
    {
      "field": "biochemistry",
      "origin": "biorxiv:molecular biology",
      "subfield": "molecular-biology"
    }
  ],
  "title": "A dedicated motif in human polymerase gamma enables DNA synthesis through replication roadblocks",
  "title_text": "A dedicated motif in human polymerase gamma enables DNA synthesis through replication roadblocks",
  "type": "preprint",
  "updated_at": "2026-10-30T12:29:07Z",
  "url": "https://research.draconitic.com/paper/d3j6nn4g5zy1m",
  "venue": "bioRxiv",
  "versions": []
}