Biochemical and Structural Characterization of Fapy·dG Replication by Human DNA Polymerase β

N6-(2-deoxy-,{beta}-D-erythro-pentofuranosyl)-2,6-diamino-4-hydroxy-5-formamido-pyrimidine (Fapy*dG) is formed from a common intermediate and in comparable amounts to the well-studied mutagenic DNA lesion 8-oxo-7,8-dihydro-2-deoxyguanosine (8-OxodGuo). Fapy*dG preferentially gives rise to G [->] T transversions and G [->] A transitions. However, the molecular basis by which Fapy*dG is processed by DNA polymerases during this mutagenic process remains poorly understood. To address this we investigated how DNA polymerase {beta} (Pol {beta}), a model mammalian polymerase, bypasses a templating Fapy*dG, inserts Fapy*dGTP, and extends from Fapy*dG at the primer terminus. When Fapy*dG is present in the template, Pol {beta} incorporates TMP less efficiently than either dCMP or dAMP. Kinetic analysis revealed that Fapy*dGTP is a poor substrate but is incorporated [~]3-times more efficiently opposite dA than dC. Extension from Fapy*dG at the 3-terminus of a nascent primer is inefficient due to the primer terminus being poorly positioned for catalysis. Together these data indicate that mutagenic bypass of Fapy*dG is likely to be the source of the mutagenic effects of the lesion and not Fapy*dGTP. These experiments increase our understanding of the promutagenic effects of Fapy*dG.