title: 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.
authors.affiliation: †Departments of Chemistry and Toxicology, University of Guelph, Guelph, ON, Canada N1G 2W1.
authors.forename: Aaron A
authors.initials: AA
authors.lastname: Witham
authors.affiliation: †Departments of Chemistry and Toxicology, University of Guelph, Guelph, ON, Canada N1G 2W1.
authors.forename: Anne M R
authors.initials: AM
authors.lastname: Verwey
authors.affiliation: †Departments of Chemistry and Toxicology, University of Guelph, Guelph, ON, Canada N1G 2W1.
authors.forename: Michael
authors.initials: M
authors.lastname: Sproviero
authors.affiliation: †Departments of Chemistry and Toxicology, University of Guelph, Guelph, ON, Canada N1G 2W1.
authors.forename: Richard A
authors.initials: RA
authors.lastname: Manderville
authors.affiliation: ‡Department of Chemistry and Biochemistry, University of Lethbridge, Lethbridge, Alberta, Canada T1K 3M4.
authors.forename: Purshotam
authors.initials: P
authors.lastname: Sharma
authors.affiliation: ‡Department of Chemistry and Biochemistry, University of Lethbridge, Lethbridge, Alberta, Canada T1K 3M4.
authors.forename: Stacey D
authors.initials: SD
authors.lastname: Wetmore
mesh_terms: D002713:Chlorine / Q000737:chemistry / Q000378:metabolism*; D018736:DNA Adducts / Q000737:chemistry / Q000378:metabolism*; D004259:DNA-Directed DNA Polymerase / Q000145:classification / Q000378:metabolism*; D006147:Guanine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D015394:Molecular Structure
chemical_list: D018736:DNA Adducts; D002713:Chlorine; D006147:Guanine; D004259:DNA-Directed DNA Polymerase