A dedicated motif in human polymerase gamma enables DNA synthesis through replication roadblocks
- 1Spanish National Cancer Research Centre (CNIO), Melchor Fernandez Almagro 3, 28029, Madrid, Spain
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.
Related
- NEIL2 regulates RNA polymerase II dynamics to suppress transcription replication conflicts in pancreatic cancer
- Structure of an RNA polymerase ribozyme replication complex
- Aclarubicin disrupts RNA polymerase II progression at replication-coupled histone genes
- Polymerase iota coordinates adaptation to replication stress in BRCA2-deficient mammary epithelial cells
- Rapid, Efficient Synthesis of Double-stranded DNA Standards for Quantitative PCR-Based Detection of HSV-2 using Polymerase Cycling Assembly.
BibTeX
@misc{miguezamil2026dedicated,
title = {{A dedicated motif in human polymerase gamma enables DNA synthesis through replication roadblocks}},
author = {S. Miguez-Amil and A. Grande-Garcia and I. Plaza Garcia Abadillo and M. Moreno-Morcillo and E. Casajus-Pelegay and E. Arean-Ulloa and C. Chacon-Sanchez and G. L. Ciesielski and B. Ibarra and R. Fernandez-Leiro},
howpublished = {bioRxiv},
year = {2026},
month = sep,
doi = {10.64898/2026.09.27.754746},
url = {https://www.biorxiv.org/content/10.64898/2026.09.27.754746v1},
note = {CC BY-NC-ND},
}
RIS
TY - UNPB
TI - A dedicated motif in human polymerase gamma enables DNA synthesis through replication roadblocks
AU - S. Miguez-Amil
AU - A. Grande-Garcia
AU - I. Plaza Garcia Abadillo
AU - M. Moreno-Morcillo
AU - E. Casajus-Pelegay
AU - E. Arean-Ulloa
AU - C. Chacon-Sanchez
AU - G. L. Ciesielski
AU - B. Ibarra
AU - R. Fernandez-Leiro
PY - 2026
DA - 2026/09/27
PB - bioRxiv
DO - 10.64898/2026.09.27.754746
UR - https://www.biorxiv.org/content/10.64898/2026.09.27.754746v1
AB - 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.
ER -