Pol γ possesses separate metal binding sites for polymerase and strand displacement functions

Accurate replication of the mitochondrial genome (mtDNA) depends on DNA polymerase γ (Pol γ), yet its strand-displacement activity has been reported with varying outcomes across studies. Here we show that human Pol γ carries out robust, processive strand-displacement synthesis under physiological divalent metal-ion concentrations. We identify two functional classes of metal-binding sites: high-affinity sites that support DNA synthesis and unwinding, and low-affinity sites that selectively suppress unwinding without impairing polymerase activity. Pol γ efficiently displaces DNA/DNA duplex and RNA/DNA hybrids, supporting a role in RNA primer removal during mtDNA replication. Cryo-EM structures of Pol γ bound to fork-mimicking DNA reveal conformational states corresponding to progressive duplex unwinding and identify structural elements that facilitate strand displacement. These findings establish a metal-dependent mechanism for Pol γ activity and reconcile previous discrepancies in its reported unwinding capacity.