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Surface plasmon resonance (SPR) wasused to measure polymerase-bindinginteractions 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 usedwere exo-nuclease-deficient Klenow fragment (Kf-exo–) or polymerase β (pol β). Specific binary and ternaryDNA binding affinities of the enzymes were characterized at subnanomolarconcentrations. The SPR results showed that Kf-exo–binds strongly to a double strand/single strand template/primerjunction, whereas pol β binds preferentially to double-strandedDNA having a one-nucleotide gap. Both enzymes exhibited tight bindingto native DNA, with high nucleotide selectivity, where the KDvalues for each base pair increased in theorder dCTP ≪ dTTP ∼ dATP ≪ dGTP. In contrastto that for pol β, Kf-exo–binds tightly tolesion-modified templates; however, both polymerases exhibited minimalnucleotide selectivity toward adducted DNA. Primer steady-state kineticsand 19F NMR results support the SPR data. The relativeinsertion efficiency finsof dCTP oppositeFABP was significantly higher in the TG*A sequencecompared to that in CG*A. Although Kf-exo–was not sensitive to the presence of a DNA lesion,FAAF-induced conformational heterogeneity perturbed the active siteof pol β, weakening the enzyme’s ability to bind to FAAFadducts compared to FABP adducts. The present study demonstrates theeffectiveness of SPR for elucidating how lesion-induced conformationalheterogeneity affects the binding capability of polymerases and ultimatelythe nucleotide insertion efficiency. [ABSTRACT FROM AUTHOR]

We used surface plasmon resonance (SPR) to characterizethe bindinginteractions between the exonulease-free Klenow fragment (Kf-exo–) and unmodified and modified dG adducts derived fromarylamine carcinogens: fluorinated 2-aminofluorene (FAF), 2-acetylaminofluorene(FAAF), and 4-aminobiphenyl (FABP). Tight polymerase binding was detectedwith unmodified dG and the correct dCTP. The discrimination of correctversus incorrect nucleotides was pronounced with KDvalues in the order of dCTP ≪ dTTP < dATP< dGTP. In contrast, minimal selectivity was observed for the modifiedtemplates with Kf-exo–binding tighter to the FAAF(koff: 0.02 s–1) andFABP (koff: 0.01 s–1) lesions than to FAF (koff: 0.04 s–1). [ABSTRACT FROM AUTHOR]

Arylamine modification of guanine base at the G 3 position in the Nar I sequence (-G 1 G 2 CG 3 CC-) causes a frameshift mutation. Polymerase and 19 F NMR studies have shown that the next flanking base at the 3' position to the G 3 adduct modulates the mutational outcome because of its different conformations. Here, we have studied the interaction of the 16-mer Nar I sequence (5'-CTCTCG 1 G 2 C G 3 CXATCAC-3') (G 3 = N -acetyl-2-aminofluorene (AAF)-dG and X is either C or T) with [Ru(phen) 2 (dppz)] 2+ (phen = 1,10-phenanthroline and dppz = dipyrido[3,2- a :2',3'- c ]phenazine). Interaction studies between isomers of Ru(II) and two oligonucleotide models, viz., (a) full duplex, and (b) slipped mutagenic intermediate (SMI), have been carried out. Luminescence studies reveal that the sensitivity of Ru(II) with an adduct increases 2- to 3-fold compared to that of control in full duplex. In SMI, the sensitivity of Ru(II) varies with the next flanking base and in the order of -G AAF CC > -G AAF CT. Microscale thermophoretic data reveal that in full duplex Λ-Ru binds to -G AAF CT- by 13- and 4-fold stronger than its control and -G AAF CC-, respectively. In SMI, Δ-Ru binds to -G AAF CC- (41% stacked ( S ) conformer) by 3-fold while -G AAF CT- (86% major groove ( B ) conformer) weakens the binding of Λ-Ru by 250-fold compared to the control. The results presented here reveal that the binding of Ru(II) not only depends on conformations of the AAF-dG adduct but also is isomer-centric and might be helpful in determining the conformational heterogeneity of other covalent aryl/heterocyclic amine-DNA adducts.