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

Abstract

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 19 F NMR.

Energy Landscapes for Base-Flipping in a Model DNA Duplex

“…How this enzyme knows the history of the nucleobase it interacts with is unknown. 42 A recent F-NMR study has shown that there may be a link between the sequence that is more prone to methylation, and a similar sequence that has been observed to interact favorably with TDG. 42 In any case, the enzyme differentiates between T in GT from the T in AT.…”

“… 42 A recent F-NMR study has shown that there may be a link between the sequence that is more prone to methylation, and a similar sequence that has been observed to interact favorably with TDG. 42 In any case, the enzyme differentiates between T in GT from the T in AT. This observation suggests an important role for the WC partner of the base being flipped out.…”

Inequality in the Frequency of the Open States Occurrence Depends on Single 2H/1H Replacement in DNA

“…All described fluctuations of n max and n min after single 2 H/ 1 H replacement can result in DNA damage due to the slowdown in the rate of bubble forming (especially non-superhelical stress-induced denaturation bubbles having more than 4 base-pairs), including the promoter regions and binding points of specific proteins (e.g., DNA repair enzymes [41,55]), or because of the overwhelming rate of flipping-out DNA nucleobases that can be accompanied by increasing both the speed of modification of their chemical structure and mismatched protein-DNA interaction [56][57][58][59].…”

Atomic resolution of short-range sliding dynamics of thymine DNA glycosylase along DNA minor-groove for lesion recognition

“…Notably, the relatively high HB occupancy observed in S8 is in fact attributed to two adjacent G·C bps. The above results may provide the molecular explanations for the fact that TDG tends to target to the CpG-riched region ( 82 , 83 ).…”