Chlorine Functionalization of a Model Phenolic C8-Guanine Adduct Increases Conformational Rigidity and Blocks Extension by a Y-Family DNA Polymerase

Abstract

Certain phenoxyl radicals can attach covalently to the C8-site of 2'-deoxyguanosine (dG) to afford oxygen-linked C8-dG adducts. Such O-linked adducts can be chemically synthesized through a nucleophilic displacement reaction between a phenolate and a suitably protected 8-Br-dG derivative. This permits the generation of model O-linked C8-dG adducts on scales suitable for insertion into oligonucleotide substrates using solid-phase DNA synthesis. Variation of the C8-aryl moiety provides an opportunity to derive structure-activity relationships on adduct conformation in duplex DNA and replication bypass by DNA polymerases. In the current study, the influence of chlorine C8-dG functionalization on in vitro DNA replication by Klenow fragment exo(-) (Kf(-)) and the Y-family polymerase (Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4)) has been determined. Model O-linked C8-dG adducts derived from the pentachlorophenoxyl radical ([PCP]G) and 2,4,6-trichlorophenoxyl radical ([TCP]G) were inserted into the reiterated G3-position of the NarI sequence (12-mer, NarI(12); and 22-mer, NarI(22)), which is a known hotspot for frameshift mutations mediated by N-linked polycyclic C8-dG adducts in bacterial mutagenesis. Within the NarI(12) duplex, the unsubstituted C8-phenoxy-dG ([PhO]G) adduct adopts a minimally perturbed B-form helix. Chlorination of [PhO]G to afford [PCP]G does not significantly change the adduct conformation within the NarI(12) duplex, as predicted by molecular dynamics simulations. However, when using NarI(22) for DNA synthesis in vitro, the chlorinated [PCP]G and [TCP]G lesions significantly block DNA replication by Kf(-) and Dpo4, whereas [PhO]G is readily bypassed. These findings highlight the impact that chlorine substituents impart to bulky C8-dG lesions.

C-Linked 8-aryl guanine nucleobase adducts: biological outcomes and utility as fluorescent probes

“…For example, O-linked adducts have been connected with pentachlorophenol (PCP, Fig. 1b ) found in pesticides, disinfectants and wood preservatives, 22 , 23 , 25 , 27 while the food mutagen ochratoxin A (OTA) arising from several species of ( Aspergillus and Penicillium ) fungi has been linked to the formation of a C-linked adduct ( Fig. 1b ).…”

Adduct Fluorescence as a Tool to Decipher Sequence Impact on Frameshift Mutations Mediated by a C-Linked C8-Biphenyl-Guanine Lesion

“…The aryl ring may be directly attached to the C8-site of dG to afford C-linked varieties that are produced by the phenolic food toxin ochratoxin A (OTA), − the polycyclic aromatic hydrocarbon (PAH) benzo­[ a ]­pyrene, or carcinogenic aryl hydrazines. , Alternatively, the aryl group and dG nucleobase may be separated by a flexible N- or O-linked tether. The N-linked C8-dG adducts are produced by arylamine and heterocyclic aromatic amine carcinogens, − while the O-linked C8-dG adducts are produced by chlorophenolic toxins, such as pentachlorophenol. − Among the multiple factors that dictate the relationship between adduct formation and mutagenesis, the combination of adduct structure and sequence context is a critical chemical basis of how enzymes involved in the DNA damage response and replication cope with these changes.…”

“…Our interest in C8-dG adducts has focused on the C- and O-linked analogs , in which a variety of C- and O-linked C8-dG adducts have been inserted into the G 3 position of Nar I to determine the impact of linkage type on adduct conformation and in vitro mutagenicity using primer extension assays. ,− Bulky O- and C-linked C8-dG adducts strongly stall primer extension by Kf – at the n + 1 site. The correct base C is mainly inserted opposite the lesion, along with small amounts of A. ,, In most instances, full-length extension products are not observed, and the GC dinucleotide deletion product has never been detected. These observations appear to mimic the results of synthesis by Kf – over AAF-dG at non-iterated positions .…”

Mutagenicity of Ochratoxin A: Role for a Carbon-Linked C8–Deoxyguanosine Adduct?

“…The OT–dG lesion containing a direct C8–C linkage (denoted C-linked C8–dG adduct) is a fairly common adduct type, with carcinogenic aryl hydrazines, , polycyclic aromatic hydrocarbons (PAHs), estrogens, , and nitroaromatics all producing C-linked C8–dG adducts following metabolic activation. Other C8–dG adducts include N-linked derivatives produced by arylamine carcinogens − and O-linked C8–dG adducts produced by phenolic toxins. − For the N-linked adducts, a variety of common conformational preferences of the associated damaged DNA duplex have been well characterized, that is, the major groove (B-type), the base-displaced stacked or intercalated (S-type), and the minor groove wedge (W-type) conformations (Figure ). , Furthermore, N-linked C8–dG adducts that exhibit potent mutagenicity possess polycyclic aromatic structures and, despite conformational heterogeneity, favor the syn conformation that generates S-type or W-type duplexes. , On the other hand, the single-ringed N-linked C8-aniline–dG adduct favors the B-type conformation and lacks potent mutagenicity .…”

“… , Furthermore, N-linked C8–dG adducts that exhibit potent mutagenicity possess polycyclic aromatic structures and, despite conformational heterogeneity, favor the syn conformation that generates S-type or W-type duplexes. , On the other hand, the single-ringed N-linked C8-aniline–dG adduct favors the B-type conformation and lacks potent mutagenicity . The unsubstituted phenolic O-linked C8–dG adduct also favors the B-type conformation and is replicated as a natural G. , Although the N-linked and O-linked C8–dG adducts both contain a flexible (amine or ether) tether separating the dG component from the aryl ring, the bulky moiety of C-linked adducts is closer to the damaged nucleobase, and the conformational preferences and biological outcomes are less understood.…”