A guardian residue hinders insertion of a Fapy•dGTP analog by modulating the open-closed DNA polymerase transition

4,6-Diamino-5-formamidopyrimidine (Fapy center dot dG) is an abundant form of oxidative DNA damage that is mutagenic and contributes to the pathogenesis of human disease. When Fapy center dot dG is in its nucleotide triphosphate form, Fapy center dot dGTP, it is inefficiently cleansed from the nucleotide pool by the responsible enzyme in Escherichia coli MutT and its mammalian homolog MTH1. Therefore, under oxidative stress conditions, Fapy center dot dGTP could become a pro-mutagenic substrate for insertion into the genome by DNA polymerases. Here, we evaluated insertion kinetics and high-resolution ternary complex crystal structures of a configurationally stable Fapy center dot dGTP analog, -C-Fapy center dot dGTP, with DNA polymerase . The crystallographic snapshots and kinetic data indicate that binding of -C-Fapy center dot dGTP impedes enzyme closure, thus hindering insertion. The structures reveal that an active site residue, Asp276, positions -C-Fapy center dot dGTP so that it distorts the geometry of critical catalytic atoms. Removal of this guardian side chain permits enzyme closure and increases the efficiency of -C-Fapy center dot dG insertion opposite dC. These results highlight the stringent requirements necessary to achieve a closed DNA polymerase active site poised for efficient nucleotide incorporation and illustrate how DNA polymerase has evolved to hinder Fapy center dot dGTP insertion.