Defining the Role of Nucleotide Flipping in Enzyme Specificity Using ¹⁹F NMR

A broad range of proteins employ nucleotide flipping to recognize specific sites in nucleic acids, including DNA glycosylases, which remove modified nucleobases to initiate base excision repair. Deamination, a pervasive mode of damage, typically generates lesions that are recognized by glycosylases as being foreign to DNA. However, deamination of 5-methylcytosine (mC) generates thymine, a canonical DNA base, presenting a challenge for damage recognition. Nevertheless, repair of mC deamination is important because the resulting G·T mispairs cause C → T transition mutations, and mC is abundant in all three domains of life. Countering this threat are three types of glycosylases that excise thymine from G·T mispairs, including thymine DNA glycosylase (TDG). These enzymes must minimize excision of thymine that is not generated by mC deamination, in A·T pairs and in polymerase-generated G·T mispairs. TDG preferentially removes thymine from DNA contexts in which cytosine methylation is prevalent, including CG and one non-CG site. This remarkable context specificity could be attained through modulation of nucleotide flipping, a reversible step that precedes base excision. We tested this idea using fluorine NMR and DNA containing 2'-fluoro-substituted nucleotides. We find that dT nucleotide flipping depends on DNA context and is efficient only in contexts known to feature cytosine methylation. We also show that a conserved Ala residue limits thymine excision by hindering nucleotide flipping. A linear free energy correlation reveals that TDG attains context specificity for thymine excision through modulation of nucleotide flipping. Our results provide a framework for characterizing nucleotide flipping in nucleic acids using ¹⁹F NMR.