A steric gate prevents mutagenic dATP incorporation opposite 8-oxo-deoxyguanosine in mitochondrial DNA polymerases

Reactive oxygen species (ROS) generate DNA lesions that alter genome integrity. Among those DNA lesions, 7,8-dihydro-8-oxo-2′-deoxyguanosine (8-oxodG) is particularly mutagenic. 8-oxodG efficiently incorporates deoxycytidine monophosphate (dCMP) and deoxyadenosine monophosphate (dAMP) via base pairing mediated by its anti and syn conformations, respectively. In family-A DNA polymerases (DNAPs), the amino acids responsible for modulating dCMP or dAMP incorporation across 8-oxodG are located in a determined structural position. Those residues are a conserved tyrosine located at the N terminus of the α-helix O and a nonconserved residue located six amino acids after this conserved tyrosine. In yeast mitochondrial DNAP (DNA-directed DNA polymerase gamma MIP1 [Mip1]), those residues correspond to amino acids Y757 and F763. We hypothesized that the phenyl group of the F763 residue impinges on the syn conformation of 8-oxodG, therefore reducing dAMP misincorporation. Here, we measured dCMP and dAMP incorporation across 8-oxodG using wild-type and F763 Mip1 mutants. Our data suggest that both residue F763 and the universally conserved Y757 assemble a steric gate that obtrudes the 8-oxodG(syn) conformation. As the human orthologue of Mip1, DNA polymerase gamma (HsPolγ) or DNAP γ, also harbors phenylalanine at the corresponding position to Mip1-F763, the steric gate mechanism might similarly be responsible for controlling HsPolγ's fidelity when tolerating 8-oxodG lesions.