A dedicated motif in human polymerase gamma enables DNA synthesis through replication roadblocks

Mitochondrial DNA maintenance is critical for cellular stability, and issues with mtDNA replication are connected to various mitochondrial disorders. While Pol{gamma} (DNA polymerase gamma) must navigate duplex junctions and stable secondary structures during replication, the way the human enzyme bypasses these obstacles is not entirely clear.

By examining cryo-electron microscopy structures of Pol{gamma} interacting with forked DNA, G-quadruplex DNA and DNA attached to mitochondrial single-stranded DNA-binding protein (mtSSB), researchers have identified a common pathway for template entry along the catcher domain regardless of the substrate and active-site structures.

Within this domain, an arginine-rich helix with R1026, R1030 and R1034 forms a Template Stabilising Motif (TSM). Experiments show that disrupting the TSM leads to impaired strand displacement, RNA-DNA hybrid displacement and synthesis through G4-forming sequences, while preserving synthesis on unstructured templates. Single-molecule optical-tweezers experiments also indicate that mechanical destabilization of the fork can restore mutant strand-displacement activity, while force or mtSSB can restore primer-extension kinetics on ssDNA templates.

These findings highlight the role of the TSM in sustaining productive template engagement and reveal template stabilization as a shared mechanism that allows Pol{gamma} to overcome structurally varied barriers in mtDNA.