Structural Influence of C8-Phenoxy-Guanine in the NarI Recognition DNA Sequence

Abstract

Phenoxyl radicals can covalently attach to the C8 site of 2'-deoxyguanosine (dG) to generate oxygen-linked biaryl ether C8-dG adducts. To assess the structural impact of an O-linked C8-dG adduct in duplex DNA, C8-phenoxy-G ((PhO)G) and C8-4-fluorophenoxy-G ((4FPhO)G) were incorporated into the G(3) position of the 12-mer NarI recognition sequence (5'-CTCGGCXCCATC, where X = G, (PhO)G, or (4FPhO)G) using solid-phase DNA synthesis with O-linked C8-dG phosphoramidites. The modified strands were hybridized to six different complementary strands that include regular base pairing to C [NarI'(C)], mismatches with G, A, T [NarI'(N)], and an abasic site [NarI'(THF)], and a 10-mer sequence to model a -2 deletion duplex [NarI'(-2)]. All duplex structures were characterized using UV-vis thermal melting temperature analysis, and in each instance, the O-linked C8-phenoxy-G adducts were found to destabilize the duplex relative to the unmodified controls. The most stable duplex structures match the O-linked C8-dG adduct against C and a G mismatch, which are comparable in terms of stability. These duplexes were further characterized using circular dichroism, dynamic (19)F nuclear magnetic resonance experiments, and molecular dynamics simulations. On the basis of these findings, (PhO)dG adopts the B conformation opposite C, with the phenoxy moiety residing in the solvent-exposed major groove. However, opposite the G mismatch, (PhO)dG adopts a "W-type" wedge conformation with the phenoxy group residing in the minor groove. These studies predict that the O-linked C8-dG lesion (PhO)G will have a weak mutagenic effect, as determined for the corresponding single-ringed nitrogen-linked C8-dG adduct derived from aniline.

Mutagenicity Analysis of C8-Phenoxy-Guanine in the NarI Recognition DNA Sequence

“…Such stalling in vivo is believed to be a trigger for the recruitment of Y-family translesion polymerases [26], which have a larger solvent-exposed active site to accommodate bulky DNA lesions for more probable effective bypass [27]. Mutagenesis experiments in vitro support our predictions derived from structural analysis of the PhOdG lesion within NarI(12) [23], and suggest that the O-linked C8-dG adduct is unlikely to contribute strongly to phenol toxicity.…”

“…These findings correlated with the conformational preference for the corresponding single-ringed N-linked C8-dG adduct produced by aniline [24]. On the basis of this comparison, the PhOdG lesion was predicted to be weakly mutagenic [23].…”

“…Full synthetic details of the PhOdG phosphoramidite have been previously published [23]. The 15-mer primer oligonucleotide was purchased from Sigma Genosys (Oakville, ON, Canada) and was purified by Sigma using polyacrylamide gel electrophoresis (PAGE).…”

“…We recently incorporated C8-phenoxy-2′-deoxyguanine (PhOdG) into the G 3 position of the NarI recognition sequence within a 12-mer oligonucleotide (5′-CTCGGCXCCATC, G 3 = X = PhOdG) in order to study its structural characteristics to predict mutagenic outcome [23]. The NarI sequence is a hot-spot for frameshift mutations mediated by the N-linked C8-dG adduct of N-acetyl-2-aminofluorene (AAF) [19][20][21].…”

“…The NarI sequence is a hot-spot for frameshift mutations mediated by the N-linked C8-dG adduct of N-acetyl-2-aminofluorene (AAF) [19][20][21]. Within the NarI(12) duplex, the PhOdG lesion was shown to adopt the major groove B conformation opposite C, with minimal disruption to the duplex structure [23]. These findings correlated with the conformational preference for the corresponding single-ringed N-linked C8-dG adduct produced by aniline [24].…”