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)).
Department of Biomedical and Pharmaceutical Sciences, College of Pharmacy, University of Rhode Island, Kingston, RI 02881, USA.
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D015073:2-Acetylaminofluorene / Q000737:chemistry*; D000611:Aminobiphenyl Compounds / Q000737:chemistry*; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D018736:DNA Adducts / Q000737:chemistry; D004256:DNA Polymerase I / Q000378:metabolism*; D003838:Deoxyadenine Nucleotides / Q000737:chemistry; D003845:Deoxycytosine Nucleotides / Q000737:chemistry; D003848:Deoxyguanine Nucleotides / Q000737:chemistry; D005449:Fluorenes / Q000737:chemistry*; D007700:Kinetics; D020349:Surface Plasmon Resonance; D013942:Thymine Nucleotides / Q000737:chemistry
D000611:Aminobiphenyl Compounds; D018736:DNA Adducts; D003838:Deoxyadenine Nucleotides; D003845:Deoxycytosine Nucleotides; D003848:Deoxyguanine Nucleotides; D005449:Fluorenes; D013942:Thymine Nucleotides; C006757:4-biphenylamine; C024107:2'-deoxycytidine 5'-triphosphate; C012177:2-aminofluorene; C029603:deoxyguanosine triphosphate; D004247:DNA; D015073:2-Acetylaminofluorene; D004256:DNA Polymerase I; C026600:2'-deoxyadenosine triphosphate; C024157:thymidine 5'-triphosphate