Structures of 2-Acetylaminofluorene Modified DNA Revisited: Insight into Conformational Heterogeneity

Abstract

Despite the extensive data on dG-AAF, the major DNA adduct derived from the model carcinogen 2-acetylaminofluorene, little is known with respect to its solution structures. Here we provide NMR/CD evidence for three conformers of dG-AAF in duplex DNA: major groove (B), basedisplaced stacked (S), and minor groove wedge (W). The S/B/W-conformational heterogeneities were found to be sensitive to the nature of the flanking DNA sequence contexts and pH.Arylamines and their nitro derivatives are a major group of environmental mutagens, and have been implicated in the etiology of human cancers (1). In vivo metabolic activation of the prototype carcinogens 2-acetylaminofluorene (2) and 2-nitrofluorene (3) results in production of dG-AF1 and dG-AAF as major stable adducts (Fig. 1a) (4). Despite their structural similarities, the two DNA adducts exhibit distinct mutation and repair activities. dG-AF has been shown to adopt multiple conformers, giving rise to unique sequencedependent mutation and repair outcomes (5,6). However, very little is known about the structure-function relationships of dG-AAF, which is remarkable given the extensive in vivo and in vitro data that have accumulated related to AAF (3,277 items on PubMed Search for "acetylaminofluorene").In 1993, O'Handley et al. (7) reported that ~70% of an AAF-modified 9-mer duplex in the CG[AAF]C sequence context adopts a base-displaced S-conformer with stacking of the fluorene moiety into a double helix (Fig. 1c). The structure of the remaining 30% of the AAF-modified duplex has not been elucidated. Using an 19 F NMR approach (8), we showed that a 12-mer duplex in the TG*A sequence context (Duplex II, G*=FAAF, Fig. 1b) exhibits 40:60% 19 F signals with an exclusive presence of the S-conformer, differing only in the Nacetyl group's cis and trans conformation (9). This result contrasts with data from AF and FAF (Fig. 1a), which consistently adopt an S/B conformational heterogeneity (6,10).We here present new data that provides further insights on the conformers that contribute to the conformational mix of dG-AAF in solution. Specifically, our results are consistent with adoption by dG-AAF of a major groove B-type (B) and a minor groove "wedge" (W) conformer, in addition to the S-conformer elucidated by O'Handley et al. (7) (Fig. 1c), and that sequence context determines the population balance between the states. The B-type conformer has a glycosyl bond conformation that is anti, while S and W are syn. The (11) and Pol iota (12). As observed in the S-conformer, the AAF-modified dG adopts the syn conformation; however, instead of having disrupted hydrogen bonding, the modified dG can pair with the complementary dC utilizing its Hoogsteen edge; this places the acetylamino moiety in the narrow minor groove (13) (Fig. 1c). Although this "wedge" W-conformer led to severe distortion of the DNA binding area in the active site of Pol iota (12), it is a distinct possibility in polymerase-free duplex DNA (13). NIH Public AccessIn the present study, we conducted spectr...

Insights into the Conformation of Aminofluorene-Deoxyguanine Adduct in a DNA Polymerase Active Site

“…The same trend was observed with various fully paired and deletion duplexes modified with the bulky N-acetylated FAF analog that specifically revealed a mixture of B, S, and W conformations (Fig. 1C) in the Ϫ115.0 ϳ Ϫ115.5 ppm, Ϫ115.5 ϳ Ϫ117.0 ppm, and Ϫ116.5 ϳ Ϫ118.0 ppm ranges, respectively (38). Accordingly, the signals in Fig.…”

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

“…16, 29 Consequently, we assigned the −116.0 and −118.1 ppm signals at 5°C of the fully paired duplex as the S- and W-conformations, respectively (Fig. 8a).…”

“…14, 15, 17, 19 Additionally, the bulky N -acetylated AAF adopts a W conformer, in which the carcinogen is placed in the narrow minor groove. 16 The relative population balance of these conformers is governed primarily by the nature of the base sequences surrounding the lesion. The resulting conformational heterogeneity has been shown to be associated with different mutational and repair outcomes.…”

“…14–19 On the other hand, the bulky N -acetylated dG-AAF lesion exists primarily in the highly distorting syn -glycosidic stacked (S)- or wedge (W)-conformations and strongly blocks the polymerase, thus resulting in frameshift mutations. 16–18, 20 In mammalian cells, however, the two lesions mostly result in point mutations. 21–23 …”

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

“…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.…”