Identifying the Molecular Mechanisms of Thymine DNA Glycosylase (TDG) Substrate Specificity

Abstract

Thymine DNA glycosylase (TDG) helps maintain genomic integrity by removing thymine from G·T mispairs arising via deamination of 5-methylcytosine (mC). TDG employs strict regulation for both the opposing guanine, as well as the base downstream of the target thymine, in order to limit removal of thymine from canonical A·T pairs, as erroneous removal of thymine from A·T pairs is mutagenic and cytotoxic. TDG also excises 5-formylcytosine (fC) and 5-carboxylcytosine (caC), oxidation products of mC generated by ten-eleven translocation (TET) enzymes during active DNA demethylation. Remarkably, using single-turnover kinetics reactions to determine the maximum rate of substrate removal, k¬max, we find that TDG activity for fC and caC shows little dependence on the opposing base or the downstream base, revealing a major difference in specificity for excision of fC and caC relative to T. Using a novel 19F NMR approach to determine the flipping equilibrium for thymine into the TDG active site, we establish that specificity during thymine excision manifests largely by modulating the stability for thymine flipping in the active site. Structure-function analysis employing a variety of opposing bases reveals that both the thermodynamic stability (ΔH) of A·T pairs, as well as direct contacts between TDG and the opposing base, contribute to opposing base specificity. The differences in specificity observed for thymine versus fC or caC are likely explained by interactions between these substrates and the TDG active site. Structural information obtained from x-ray crystallography, combined with TDG mutational studies, identified several TDG active site residues that form stabilizing interactions with fC and caC, helping to both stabilize base flipping into the active site, as well as enhance the chemical steps of base excision. Conversely, two conserved residues in TDG, A145 and H151, limit stability of thymine in the active site, and destabilize thymine as a leaving group. As a result, additional contacts between both the opposing guanine, as well as the guanine downstream of the target thymine, appear necessary to orient thymine in a manner which produces stable, productive flipping into the active site.