title: Probing the sequence effects on NarI-induced -2 frameshift mutagenesis by dynamic 19F NMR, UV, and CD spectroscopy.
abstract: The NarI recognition sequence (5'-G1G2CG3CN-3') is the most vulnerable hot spot for frameshift mutagenesis induced by the carcinogen 2-aminofluorene and its analogues in Escherichia coli. Lesioning of the guanine in the G3 position induces an especially high frequency of -2 deletion mutations; vulnerability to these mutations is modulated by the nature of the nucleotide in the N position (C approximately A > G > T). The objective of the present study was to probe the structural basis of this N-mediated influence on the propensity of the G3 lesion to form a slipped mutagenic intermediate (SMI) during translesion synthesis. We studied NarI-based fully paired [(5'-CTCG1G2CG3*CNATC-3')(5'-GATNCGGCCGAG-3'), N = dC or dT] and -2 deletion [(5'-CTCG1G2CG3*CNATC-3')(5'-GATNGCCGAG-3'), N = dC or dT] duplexes, in which G* was either AF [N-(2'-deoxyguanosin-8-yl)-2-aminofluorene] or the 19F probe FAF [N-(2'-deoxyguanosin-8-yl)-7-fluoro-2-aminofluorene]. The latter sequences mimic the bulged SMI for -2 deletion mutations. Dynamic 19F NMR, circular dichroism, and UV melting results indicated that the NarI-dC/-2 deletion duplex adopts exclusively an intercalated conformer, whereas the NarI-dT/-2 deletion duplex exists as multiple conformers. The data support the presence of a putative equilibrium between a carcinogen-intercalated and a carcinogen-exposed SMI for the dT/-2 duplex. A similar dT-induced conformational heterogeneity was observed for the fully paired duplexes in which all three guanines were individually modified by AF or FAF. The frequency of the carcinogen stacked S-conformation was found to be highest (69-75%) at the G3 hot spot in NarI-dC duplexes. Taken together, our results support the hypothesis that the conformational stability of the SMI is a critical determinant for the efficacy of -2 frameshift mutagenesis in the NarI sequence. We also provide evidence for AF/FAF conformational compatibility in the NarI sequences.
authors.affiliation: Department of Biomedical and Pharmaceutical Sciences, College of Pharmacy, University of Rhode Island, Kingston, Rhode Island 02881, USA.
authors.forename: Nidhi
authors.initials: N
authors.lastname: Jain
authors.forename: Yuyuan
authors.initials: Y
authors.lastname: Li
authors.forename: Li
authors.initials: L
authors.lastname: Zhang
authors.forename: Srinivasa R
authors.initials: SR
authors.lastname: Meneni
authors.forename: Bongsup P
authors.initials: BP
authors.lastname: Cho
mesh_terms: D015073:2-Acetylaminofluorene / Q000031:analogs & derivatives / Q000737:chemistry; D001483:Base Sequence; D002942:Circular Dichroism; D018736:DNA Adducts / Q000737:chemistry*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism*; D005449:Fluorenes / Q000737:chemistry*; D005461:Fluorine / Q000737:chemistry; D016368:Frameshift Mutation; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D019906:Nuclear Magnetic Resonance, Biomolecular; D009690:Nucleic Acid Conformation; D013056:Spectrophotometry, Ultraviolet
chemical_list: C052668:2-aminofluorene-DNA complex; D018736:DNA Adducts; D005449:Fluorenes; D005461:Fluorine; D015073:2-Acetylaminofluorene; D015252:Deoxyribonucleases, Type II Site-Specific; C046719:GGCGCC-specific type II deoxyribonucleases