Binary and Ternary Binding Affinities between Exonuclease-Deficient Klenow Fragment (Kf-exo^–) and Various Arylamine DNA Lesions Characterized by Surface Plasmon Resonance

We used surface plasmon resonance (SPR) to characterize the binding interactions between the exonulease-free Klenow fragment (Kf-exo^–) and unmodified and modified dG adducts derived from arylamine carcinogens: fluorinated 2-aminofluorene (FAF), 2-acetylaminofluorene (FAAF), and 4-aminobiphenyl (FABP). Tight polymerase binding was detected with unmodified dG and the correct dCTP. The discrimination of correct versus incorrect nucleotides was pronounced with K_D values in the order of dCTP ≪ dTTP < dATP < dGTP. In contrast, minimal selectivity was observed for the modified templates with Kf-exo^– binding tighter to the FAAF (k_off: 0.02 s^–1) and FABP (k_off: 0.01 s^–1) lesions than to FAF (k_off: 0.04 s^–1).