Real-Time Surface Plasmon Resonance Study of Biomolecular Interactions between Polymerase and Bulky Mutagenic DNA Lesions

Surface plasmon resonance (SPR) was used to measure polymerase-binding interactions of the bulky mutagenic DNA lesions N-(2′-deoxyguanosin-8-yl)-4′-fluoro-4-aminobiphenyl (FABP) or N-(2′-deoxyguanosin-8-yl)-7-fluoro-2-acetylaminofluorene (FAAF) in the context of two unique 5′-flanking bases (CG*A and TG*A). The enzymes used were exo-nuclease-deficient Klenow fragment (Kf-exo–) or polymerase β (pol β). Specific binary and ternary DNA binding affinities of the enzymes were characterized at subnanomolar concentrations. The SPR results showed that Kf-exo– binds strongly to a double strand/single strand template/primer junction, whereas pol β binds preferentially to double-stranded DNA having a one-nucleotide gap. Both enzymes exhibited tight binding to native DNA, with high nucleotide selectivity, where the KD values for each base pair increased in the order dCTP ≪ dTTP ∼ dATP ≪ dGTP. In contrast to that for pol β, Kf-exo– binds tightly to lesion-modified templates; however, both polymerases exhibited minimal...