Biochemical and Structural Characterization of Fapy•dG Replication by Human DNA Polymerase Β

N6-(2-deoxy-alpha,beta-d-erythro-pentofuranosyl)-2,6-diamino-4-hydroxy-5-formamido-pyrimidine (Fapy center dot 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 center dot dG preferentially gives rise to G -> T transversions and G -> A transitions. However, the molecular basis by which Fapy center dot 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 center dot dG, inserts Fapy center dot dGTP, and extends from Fapy center dot dG at the primer terminus. When Fapy center dot dG is present in the template, Pol beta incorporates TMP less efficiently than either dCMP or dAMP. Kinetic analysis revealed that Fapy center dot dGTP is a poor substrate but is incorporated similar to 3-times more efficiently opposite dA than dC. Extension from Fapy center dot 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 center dot dG is likely to be the source of the mutagenic effects of the lesion and not Fapy center dot dGTP. These experiments increase our understanding of the promutagenic effects of Fapy center dot dG. Graphical Abstract