Our genome is constantly under attack from endogenous and environmental factors like UV radiation and DNA-damaging carcinogenic chemicals. The identities of the bases immediately flanking a DNA lesion, known as the sequence context, influence how the lesion is repaired and the fidelity with which polymerases replicate past the lesion. The conformational ratio a lesion adopts, and consequently the extent of destabilization it imparts on the duplex, is highly sequence context-dependent, and ultimately determines the repair and mutational efficiencies of a lesion. Fluoroaminofluorene (FAF), is a bulky, mutagenic arylamine DNA lesion with the major adduct occurring at the C8 position of guanine (N-(2'-deoxyguanosin-8-yl)-7-fluoro-2-aminofluorene). The dG-FAF lesion is known to adopt a sequence-dependent mix of two significant conformations: major groove binding (B type) and base-displaced intercalative (S type). The conformational heterogeneity of the dG-FAF adduct in the site-specifically modified epigenetic sequence context 5′- CTTCTC#G*NCCTCATTC -3′ (where C#= cytosine or 5-methylcytosine; G*= lesion site; N= A, C, T, or G) and its effects on lesion bypass efficiency and mutagenicity were studied using fluorine-19 nuclear magnetic resonance spectroscopy (19F-NMR), UV melting, circular dichroism (CD) spectroscopy, and in vivo competitive replication of adduct bypass (CRAB) and restriction endonuclease and postlabeling (REAP) assays. Preliminary results show a flanking 5-methylcytosine (m5C) 5′ to the lesion site hinders stacking of the FAF moiety within the duplex, leading to a more thermally stable complex than in control duplexes containing canonical C as the 5′ flanking base. CRAB and REAP assays are ongoing and will give insight into how a flanking m5C affects mutagenic bypass of the dG-FAF lesion in these contexts.

Conformational heterogeneity of the fluoroaminofluorene-deoxyguanine adduct (dG-FAF) in epigenetically modified sequence contexts

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