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.

Structure and Thermodynamic Insights on Acetylaminofluorene-Modified Deletion DNA Duplexes as Models for Frameshift Mutagenesis

“…We have also accumulated an extensive collection of 19 F NMR spectra of fluorinated aminofluorene-modified duplexes in various sequence settings and found that in the base-displaced stacked (S) conformer fluorine is always shielded (upfield) relative to that of the external binding B-type conformer. 14, 15, 26, 29, 44, 48, 4919 F shielding is a hallmark for the van der Waals interaction and the ring current effect caused by the carcinogen moiety within the stacked and bulge duplexes (S-type conformation). 44 We have used a similar strategy to assign the signals in the 19 F NMR spectra of deletion duplexes, i.e., the signals (~ −115 ppm) near the coalescence temperatures arise from denatured single stranded modified exposed fluorine and non-stacked external B-type conformers, and shielded signals arise from a buried fluorinated carcinogen with excess van der Waals interactions with neighboring base pairs as occurs in S-type conformations.…”

Structural and Thermodynamic Insight into Escherichia coli UvrABC-Mediated Incision of Cluster Diacetylaminofluorene Adducts on the NarI Sequence

“…This trend is not surprising since base stacking interactions are one of the most important factors that contribute towards the thermal and thermodynamic stability of the DNA duplexes. 17, 18 …”

“…14 We also have documented that the incorporation of fluorine atom at the longest axis does not affect the overall conformational and thermodynamic profiles of aminofluorene-modified duplexes (e.g., dG-FAF, dG-FAAF). 17, 30 FAAF-modified 16-mer di-adduct oligodeoxynucleotides were prepared similarly with a longer reaction time (Figure S1, Supporting Information). 14, 15, 31 Experimental details of FAAF modification and HPLC purification are provided in the Supporting Information.…”

Unusual sequence effects on nucleotide excision repair of arylamine lesions: DNA bending/distortion as a primary recognition factor

“…UV-Melting (Cary100 Bio, Beckman) and Circular Dichroism (CD) (J-810, Jasco) experiments were performed using the previously reported procedures (7,20,23,26). …”