Understanding the Mutagenicity of O-Linked and C-Linked Guanine DNA Adducts: A Combined Experimental and Computational Approach

Abstract

The formation of DNA adducts by the attack of intermediates derived from toxic substances at C8 of 2'-deoxyguanosine (dG) is a common damaging event. Although the majority of studies on C8-dG adducts have focused on lesions containing a C8-N-C tether between the bulky moiety and the nucleobase, the formation of O-linked lesions with a similar tether topology and C-linked adducts involving direct C8-C connectivity have also been uncovered. Several studies have been done to try to better understand the structural impact and mutagenicity of O-linked and C-linked aryl C8-dG adducts, including lesions arising from unsubstituted and chloro-substituted phenols and the food mutagen ochratoxin A (OTA). Information about the structural preferences of the adducts in duplexes containing the NarI sequence has been gained from optical spectroscopy (UV-vis, CD, and fluorescence), F NMR spectroscopy, and computational chemistry (density functional theory calculations at the nucleobase, nucleoside, and nucleotide levels and molecular dynamics simulations of adducted duplexes). The replication of select adducts has also been investigated using primer-elongation assays, and model high-fidelity and Y-family polymerases. Although the (unsubstituted) O-linked phenoxy-dG adduct preferentially induces a single duplex conformation and is replicated as per natural dG, chloro substitution blocks DNA replication. In contrast, the unsubstituted C-linked phenyl-dG adduct induces mismatches, while the C-linked ortho- and para-phenoxy-dG lesions lead to conformational heterogeneity of adducted DNA indicative of strong mutagenic potential. Finally, the C-linked OTA-derived lesion exhibits the greatest conformational flexibility in duplexes, which provides structural explanations for observed outcomes in OTA-exposed cells. Overall, the variation in the conformational preferences of DNA containing O-linked and C-linked aryl-dG adducts highlights the fact that the type of C8 linkage, the presence and location of functional groups in the bulky moiety, the adduct ionization state, and the sequence context can have profound effects on the conformational outcomes of adducted DNA, which directly dictate the activity of the original toxin.

Recent Studies on DNA Adducts Resulting from Human Exposure to Tobacco Smoke

“…Computational approach. In addition to experimental techniques, computational approaches have been used to study conformational preference of the adducts in DNA, their potential to cause mismatch and mutations, and their interactions with DNA polymerases and repair enzymes [135,136,137]. For example, a multiscale computational approach demonstrated that both O 6 -POB-G and O 6 -PHB-G pair with C and T, with the latter being more mutagenic because of the difference in the bulky moiety hydrogen-bonding pattern [135].…”

Molecular Dynamics Simulations of Mismatched DNA Duplexes Associated with the Major C8-Linked 2′-Deoxyguanosine Adduct of the Food Mutagen Ochratoxin A: Influence of Opposing Base, Adduct Ionization State, and Sequence on the Structure of Damaged DNA

“…Molecular dynamics (MD) simulations on damaged DNA is a useful approach for gaining structural insights into the role of adduct formation in the associated mutagenicity. Specifically, MD simulations permit the study of DNA structural dynamics, which can be further used to explain experimental observations. − Therefore, as a first step toward understanding the impact of OTA exposure on DNA structure, we previously employed MD simulations to characterize the conformational preferences of DNA containing the OT-G adduct. , The adduct was paired against complementary cytosine in a DNA oligomer containing the Nar I recognition sequence (5′-G 1 G 2 CG 3 CC), which is the known hotspot for mutations induced by other carcinogenic C 8 -G adducts . Our analysis revealed that OTA-damaged DNA likely adopts a mixture of three distinct (i.e., the major groove (B), wedge (W), and base-displaced intercalated (S)) conformations (Figure B–D) regardless of the adduct ionization state and the lesion-site sequence context.…”

Molecular Modeling of the Major DNA Adduct Formed from Food Mutagen Ochratoxin A in NarI Two-Base Deletion Duplexes: Impact of Sequence Context and Adduct Ionization on Conformational Preference and Mutagenicity

“…Most notably, in vitro studies on animal tissues treated with OTA used 32 P–postlabeling of the DNA nucleotides to provide evidence for the formation of bulky OTA–DNA adducts, specifically with 2′-deoxyguanosine monophosphate. − Further spectroscopic studies characterized three distinct OTA-dG covalent adducts, namely C-linked C 8 –OT-dG, O-linked C 8 –OTA-dG, and N,N-linked N1,N2-OTHQ-dG . Among these lesions, the OT-dG adduct (Figure a) was found to be the most prevalent. , More recent studies have focused on understanding the mutagenicity associated with OTA. For example, an in vitro study on the Sup F gene of the mutation reporter plasmid PS189 in human Ad293 cells and the red / gam gene (Spi – ) at the renal outer medulla of rodents revealed increased mutation frequencies upon OTA exposure.…”

“…22 Among these lesions, the OT-dG adduct (Figure 1a) was found to be the most prevalent. 23,24 More recent studies have focused on understanding the mutagenicity associated with OTA. For example, an in vitro study on the SupF gene of the mutation reporter plasmid PS189 in human Ad293 cells 25 and the red/gam gene (Spi − ) at the renal outer medulla of rodents 15 revealed increased mutation frequencies upon OTA exposure.…”