ABSTRACT
N6-(2-deoxy-α,ß-d-erythro-pentofuranosyl)-2,6-diamino-4-hydroxy-5-formamido-pyrimidine (Fapyâ¢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). Fapyâ¢dG preferentially gives rise to G â T transversions and G â A transitions. However, the molecular basis by which Fapyâ¢dG is processed by DNA polymerases during this mutagenic process remains poorly understood. To address this we investigated how DNA polymerase ß (Pol ß), a model mammalian polymerase, bypasses a templating Fapyâ¢dG, inserts Fapyâ¢dGTP, and extends from Fapyâ¢dG at the primer terminus. When Fapyâ¢dG is present in the template, Pol ß incorporates TMP less efficiently than either dCMP or dAMP. Kinetic analysis revealed that Fapyâ¢dGTP is a poor substrate but is incorporated â¼3-times more efficiently opposite dA than dC. Extension from Fapyâ¢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 Fapyâ¢dG is likely to be the source of the mutagenic effects of the lesion and not Fapyâ¢dGTP. These experiments increase our understanding of the promutagenic effects of Fapyâ¢dG.
Subject(s)
DNA Polymerase beta , DNA Replication , Formamides , Furans , Pyrimidines , Humans , Crystallography, X-Ray , DNA/chemistry , DNA/metabolism , DNA Polymerase beta/metabolism , DNA Polymerase beta/chemistry , Kinetics , Models, Molecular , Pyrimidines/chemistry , Pyrimidines/metabolism , Furans/chemistry , Furans/metabolism , Formamides/metabolism , MutagenesisABSTRACT
N6-(2-deoxy-α,ß-D-erythro-pentofuranosyl)-2,6-diamino-4-hydroxy-5-formamido-pyrimidine (Fapyâ¢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). Fapyâ¢dG preferentially gives rise to G â T transversions and G â A transitions. However, the molecular basis by which Fapyâ¢dG is processed by DNA polymerases during this mutagenic process remains poorly understood. To address this we investigated how DNA polymerase ß (Pol ß), a model mammalian polymerase, bypasses a templating Fapyâ¢dG, inserts Fapyâ¢dGTP, and extends from Fapyâ¢dG at the primer terminus. When Fapyâ¢dG is present in the template, Pol ß incorporates TMP less efficiently than either dCMP or dAMP. Kinetic analysis revealed that Fapyâ¢dGTP is a poor substrate but is incorporated â¼3-times more efficiently opposite dA than dC. Extension from Fapyâ¢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 Fapyâ¢dG is likely to be the source of the mutagenic effects of the lesion and not Fapyâ¢dGTP. These experiments increase our understanding of the promutagenic effects of Fapyâ¢dG.