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Glycosylases specifically recognize and flip their target base out of the DNA helix into the enzyme's active site. Our simulations show that a partially flipped state, already present in free DNA carrying a T:G mispair, becomes the more probable state compared to the closed state after binding of thymine DNA glycosylase (TDG). Paired thymine (T:A) or methyl-cytosine (mC:G) does not exhibit a partially flipped state in free or complexed DNA. Important enzyme-DNA interactions exhibit significant strength in the intrahelical and extrahelical TDG-DNA complexes. The computed binding free energy differences suggest these interactions account for the stabilization of the partially flipped state, thereby driving the T:G mispair toward base flip. In the fully flipped state, the cognate base thymine is significantly better accommodated in the enzyme's active site than noncognate bases are, suggesting the hydrolysis step as the last of several stages at which base recognition can be achieved.

Distortions in the DNA sequence, such as damage or mispairs, are specifically recognized and processed by DNA repair enzymes. Many repair proteins and, in particular, glycosylases flip the target base out of the DNA helix into the enzyme's active site. Our molecular dynamics simulations of DNA with intact and damaged (oxidized) methyl-cytosine show that the probability of being flipped is similar for damaged and intact methyl-cytosine. However, the accessibility of the different 5-methyl groups allows direct discrimination of the oxidized forms. Hydrogen-bonded patterns that vary between methyl-cytosine forms carrying a carbonyl oxygen atom are likely to be detected by the repair enzymes and may thus help target site recognition.