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

Abstract

We used surface plasmon resonance (SPR) to characterize the binding interactions between 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 KD values in order of dCTP << dTTP < dATP < dGTP. In contrast, minimal selectivity was observed for the modified templates with Kf-exo- binding tighter to the FAAF (koff: 0.02s-1) and FABP (koff: 0.01s-1) lesions than to FAF (koff: 0.04s-1).

Insights into the Conformation of Aminofluorene-Deoxyguanine Adduct in a DNA Polymerase Active Site

“…Pol ␤ DNA Binding Kinetics-DNA polymerase binding to modified and lesion-containing DNA was monitored by SPR as described previously (34). The biotinylated DNA substrates were immobilized on streptavidin coated on a dextran surface.…”

“…Determination of Nucleotide Insertion Kinetics ParametersFor pol ␤, steady-state kinetic parameters for nucleotide incorporation opposite unmodified and FAF-modified templates were determined at 37°C as reported previously (34). Enzyme concentrations and reaction time intervals were optimized so that substrate depletion did not impact initial velocity measurements.…”

“…An 83-mer hairpin-template-primer was prepared by following a previously reported protocol (34). Briefly, a biotinylated 31-mer (optical density of 5) was annealed with a 52-mer hairpin (optical density of 6) by heating to 95°C for 5 min and cooling to room temperature.…”

“…Immobilization of Streptavidin on CM5 Chip and DNA Coating-The immobilization of streptavidin on CM5 surface was carried out using l-ethyl-3-(3-dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide amine coupling method by following the reported procedure (34). The biotinylated 84-mer DNA-hairpin with a dideoxy-terminated primer (0.25-0.3 nM) was injected for 120 -240 s over the flow cell 2 and 4 to achieve 3-4 RU through manual injection.…”

“…SPR Binding Kinetics Analysis-The surface performance, mass transport, and regeneration buffer scouting were performed as described previously (34). The binding kinetics for the interaction of pol ␤ with DNA was performed by injecting pol ␤ over the DNA surface using running buffer B (0.1 M Hepes, pH 7.4, 150 mM NaCl, 0.005% non-ionic surfactant P20, 5 mM MgCl 2 , and 100 g/ml BSA) with a flow rate of 100 l/min for 30 s followed by dissociation for 60 s. A different concentra-tion range of pol ␤ was necessary for double-stranded DNA and 1-nt gapped DNA due to the significantly different DNA binding affinities.…”