Biochemical genetics of the mitochondrial replisome

The mitochondrial replicase (pol γ ) comprises two subunits, a large catalytic core (pol γ‐α) and a smaller accessory subunit (pol γ‐β ) that enhances holoenzyme activity and processivity. Mutagenesis of four conserved sequence elements located within the spacer region between the DNA polymerase and 3′‐5′ exonuclease active sites in pol γ‐α demonstrates their functional roles in holoenzyme activity, processivity and/or DNA binding affinity. Several mutations also affect differentially DNA polymerase and exonuclease activity, and/or functional interactions with mitochondrial single‐stranded DNA‐binding protein (mtSSB). A parallel mutagenesis of human pol γ β in combination with the determination of its crystal structure and molecular modeling, elucidates the role of the accessory subunit as a novel type of processivity factor. A human pol γ /DNA complex model was developed using the structures of the pol γ β dimer and the T7 DNA polymerase ternary complex, which suggests multiple regions of subunit interaction between pol γ β and the human catalytic core that allow it to encircle the newly synthesized double‐stranded DNA, and thereby enhance DNA binding affinity and holoenzyme processivity. Functional complexes of pol γ, mtSSB and mitochondrial DNA helicase reconstitute the mitochondrial DNA replication fork. This work was supported by NIH grants GM45295 to L.S.K. and CA92584 to J.A.T.