National Center for
4FS2: Base Pairing Mechanism Of N2,3-Ethenoguanine With Dctp By Human Polymerase Iota
J. Biol. Chem. (2012) 287 p.35516-35526
N(2),3-Ethenoguanine (N(2),3-epsilonG) is one of the exocyclic DNA adducts produced by endogenous processes (e.g. lipid peroxidation) and exposure to bioactivated vinyl monomers such as vinyl chloride, which is a known human carcinogen. Existing studies exploring the miscoding potential of this lesion are quite indirect because of the lability of the glycosidic bond. We utilized a 2'-fluoro isostere approach to stabilize this lesion and synthesized oligonucleotides containing 2'-fluoro-N(2),3-epsilon-2'-deoxyarabinoguanosine to investigate the miscoding potential of N(2),3-epsilonG by Y-family human DNA polymerases (pols). In primer extension assays, pol eta and pol kappa replicated through N(2),3-epsilonG, whereas pol iota and REV1 yielded only 1-base incorporation. Steady-state kinetics revealed that dCTP incorporation is preferred opposite N(2),3-epsilonG with relative efficiencies in the order of pol kappa > REV1 > pol eta approximately pol iota, and dTTP misincorporation is the major miscoding event by all four Y-family human DNA pols. Pol iota had the highest dTTP misincorporation frequency (0.71) followed by pol eta (0.63). REV1 misincorporated dTTP and dGTP with much lower frequencies. Crystal structures of pol iota with N(2),3-epsilonG paired to dCTP and dTTP revealed Hoogsteen-like base pairing mechanisms. Two hydrogen bonds were observed in the N(2),3-epsilonG:dCTP base pair, whereas only one appears to be present in the case of the N(2),3-epsilonG:dTTP pair. Base pairing mechanisms derived from the crystal structures explain the slightly favored dCTP insertion for pol iota in steady-state kinetic analysis. Taken together, these results provide a basis for the mutagenic potential of N(2),3-epsilonG.