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

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 γ (Polγ) 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 Polγ 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 Polγ to traverse structurally diverse barriers in mtDNA.