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Thymine DNA Glycosylase Can Rapidly Excise 5-formylcytosine and 5-carboxylcytosine: Potential Implications for Active Demethylation of CpG Sites

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Thymine DNA Glycosylase Can Rapidly Excise 5-formylcytosine and 5-carboxylcytosine: Potential Implications for Active Demethylation of CpG Sites

Atanu Maiti et al. J Biol Chem.

Abstract

Thymine DNA glycosylase (TDG) excises T from G·T mispairs and is thought to initiate base excision repair (BER) of deaminated 5-methylcytosine (mC). Recent studies show that TDG, including its glycosylase activity, is essential for active DNA demethylation and embryonic development. These and other findings suggest that active demethylation could involve mC deamination by a deaminase, giving a G·T mispair followed by TDG-initiated BER. An alternative proposal is that demethylation could involve iterative oxidation of mC to 5-hydroxymethylcytosine (hmC) and then to 5-formylcytosine (fC) and 5-carboxylcytosine (caC), mediated by a Tet (ten eleven translocation) enzyme, with conversion of caC to C by a putative decarboxylase. Our previous studies suggest that TDG could excise fC and caC from DNA, which could provide another potential demethylation mechanism. We show here that TDG rapidly removes fC, with higher activity than for G·T mispairs, and has substantial caC excision activity, yet it cannot remove hmC. TDG excision of fC and caC, oxidation products of mC, is consistent with its strong specificity for excising bases from a CpG context. Our findings reveal a remarkable new aspect of specificity for TDG, inform its catalytic mechanism, and suggest that TDG could protect against fC-induced mutagenesis. The results also suggest a new potential mechanism for active DNA demethylation, involving TDG excision of Tet-produced fC (or caC) and subsequent BER. Such a mechanism obviates the need for a decarboxylase and is consistent with findings that TDG glycosylase activity is essential for active demethylation and embryonic development, as are mechanisms involving TDG excision of deaminated mC or hmC.

Figures

FIGURE 1.
FIGURE 1.
Potential mechanisms for active DNA demethylation. A, four potential mechanisms for active DNA demethylation in vertebrates are shown (AP is an abasic site). These results show that TDG can rapidly excise fC and caC, suggesting a new potential mechanism for active demethylation (larger gray arrows), involving Tet-mediated oxidation of mC, TDG excision of fC (or caC), and subsequent BER. Details regarding the previously proposed demethylation pathways (and references) are under “Introduction.” B, chemical structures for mC and its derivatives resulting from oxidation and/or deamination.
FIGURE 2.
FIGURE 2.
TDG excises fC and caC but not hmC. An electrophoretic assay of TDG activity for G·T, G·fC, G·caC, and G·hmC DNA substrates is shown. TDG (5 μm) was incubated with substrate DNA (0.5 μm) for 30 s at 37 °C followed by alkaline-induced quenching and cleavage of the DNA at TDG-generated abasic sites (S indicates substrate, and P indicates product). For G·hmC, reactions were also performed for 1 and 2 h at 37 °C and for 18 h at 22 °C.
FIGURE 3.
FIGURE 3.
Kinetics for TDG processing of G·fC, G·caC, and G·T substrates. Single turnover kinetics experiments were collected with 5 μm TDG and 0.5 μm DNA substrate at 37 °C. Fitting the data to Equation 1 provides rate constants of kmax = 2.64 ± 0.09 min−1 for G·fC (A), kmax = 0.47 ± 0.01 min−1 for G·caC (B), and kmax = 1.83 ± 0.04 min−1 for G·T (C). Data from three independent experiments are shown for each substrate.

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