Lesion Bypass of N2-Ethylguanine by Human DNA Polymerase ι

Nucleotide incorporation and extension opposite N2-ethyl-Gua by DNA polymerase ι was measured and structures of the DNA polymerase ι-N2-ethyl-Gua complex with incoming nucleotides were solved. Efficiency and fidelity of DNA polymerase ι opposite N2-ethyl-Gua was determined by steady state kinetic analysis with Mg2+ or Mn2+ as the activating metal. DNA polymerase ι incorporates dCMP opposite N2-ethyl-Gua and unadducted Gua with similar efficiencies in the presence of Mg2+ and with greater efficiencies in the presence of Mn2+. However, the fidelity of nucleotide incorporation by DNA polymerase ι opposite N2-ethyl-Gua and Gua using Mn2+ is lower relative to that using Mg2+ indicating a metal-dependent effect. DNA polymerase ι extends from the N2-ethyl-Gua:Cyt 3′ terminus more efficiently than from the Gua:Cyt base pair. Together these kinetic data indicate that the DNA polymerase ι catalyzed reaction is well suited for N2-ethyl-Gua bypass. The structure of DNA polymerase ι with N2-ethyl-Gua at the active site reveals the adducted base in the syn configuration when the correct incoming nucleotide is present. Positioning of the ethyl adduct into the major groove removes potential steric overlap between the adducted template base and the incoming dCTP. Comparing structures of DNA polymerase ι complexed with N2-ethyl-Gua and Gua at the active site suggests movements in the DNA polymerase ι polymerase-associated domain to accommodate the adduct providing direct evidence that DNA polymerase ι efficiently replicates past a minor groove DNA adduct by positioning the adducted base in the syn configuration.