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 <i>in vitro</i> 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 <i>Nar</i>I sequence (12-mer, <i>Nar</i>I­(12); and 22-mer, <i>Nar</i>I­(22)), which is a known hotspot for frameshift mutations mediated by N-linked polycyclic C8-dG adducts in bacterial mutagenesis. Within the <i>Nar</i>I­(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 <i>Nar</i>I­(12) duplex, as predicted by molecular dynamics simulations. However, when using <i>Nar</i>I­(22) for DNA synthesis <i>in vitro</i>, 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