title: Real-time surface plasmon resonance study of biomolecular interactions between polymerase and bulky mutagenic DNA lesions.
abstract: 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 nucleotide selectivity toward adducted DNA. Primer steady-state kinetics and (19)F NMR results support the SPR data. The relative insertion efficiency fins of dCTP opposite FABP was significantly higher in the TG*A sequence compared 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 site of pol β, weakening the enzyme's ability to bind to FAAF adducts compared to FABP adducts. The present study demonstrates the effectiveness of SPR for elucidating how lesion-induced conformational heterogeneity affects the binding capability of polymerases and ultimately the nucleotide insertion efficiency.
authors.affiliation: Department of Biomedical and Pharmaceutical Sciences, College of Pharmacy, University of Rhode Island , Kingston, Rhode Island 02881, United States.
authors.forename: Lifang
authors.initials: L
authors.lastname: Xu
authors.forename: V G
authors.initials: VG
authors.lastname: Vaidyanathan
authors.forename: Bongsup P
authors.initials: BP
authors.lastname: Cho
mesh_terms: D020029:Base Pairing; D001483:Base Sequence; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D018736:DNA Adducts / Q000737:chemistry; D004256:DNA Polymerase I / Q000378:metabolism*; D019951:DNA Polymerase beta / Q000378:metabolism*; D003838:Deoxyadenine Nucleotides / Q000737:chemistry; D003845:Deoxycytosine Nucleotides / Q000737:chemistry; D003848:Deoxyguanine Nucleotides / Q000737:chemistry; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D009841:Oligonucleotides / Q000032:analysis; D011485:Protein Binding; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D020349:Surface Plasmon Resonance
chemical_list: D018736:DNA Adducts; D003838:Deoxyadenine Nucleotides; D003845:Deoxycytosine Nucleotides; D003848:Deoxyguanine Nucleotides; D009841:Oligonucleotides; C024107:2'-deoxycytidine 5'-triphosphate; C029603:deoxyguanosine triphosphate; D004247:DNA; D004256:DNA Polymerase I; D019951:DNA Polymerase beta; C026600:2'-deoxyadenosine triphosphate