Research Update

Deconvoluting the Methylation/Demethylation Pathway

The importance of the process of methylation and demethylation of nucleic acids cannot be understated. This cascade of enzymatic reactions is critical to gene regulation, immune response and base repairing. To date, the mechanism for the demethylation of 5-methylcytosine (mC) has not been confirmed, though several postulated. One hypothesis involved deamination of mC to thymine, forming a G-T mismatch which is then repaired using a base excision repair (BER) enzyme.

Dr. Alexander Drohat's lab from the University of Maryland’s School of Medicine has investigated the role of thymine DNA glycosolase (TDG)¹ in demethylation. TDG may be involved in an alternative pathway involving the more recently discovered TET-proteins (TET 1-3) which convert mC to 5-hydroxymethylcytosine (5-hmC), followed by subsequent oxidation to 5-formylcytosine (5-fC) or 5-carboxycytosine (5-caC), both of which are also naturally occurring. Previous findings by the author suggest a potential role of TDG in the excision of formylcytosine and 5-hydroxycytosine. The current manuscript¹ studied the excision of 5-formyl-C and 5-carboxy-C by TDG and suggests a direct role in the crucial demethylation pathway.

To test their hypothesis, the authors prepared four synthetic oligonucleotides 28 bases in length that contained either thymine, 5-hmC, 5-fC or 5-caC at position 17. It was found that TDG can rapidly excise thymine, 5-fC and 5-caC, but was not able to excise 5-hmC at 37°C. The k_max for excision of 5-fC was 2.64 ± 0.09 min^-1, whereas the k_max for the excision of T was 1.83 ± 0.04 min^-1 and 0.47 ± 0.01 min^-1 for 5-caC.

Based on this study and their previous work, the authors concluded that an electron withdrawing group at the 5 position of cytosine is crucial for recognition by TDG. This work demonstrated that the pathway for demethylation could be oxidation of mC to 5-fC by a TET enzyme, followed by a TDG mediated BER of 5-fC to C. The authors further speculate that the specificity of TDG for the rapid excision of 5-fC, but not 5-hmC may imply a role of 5-hmC in other epigenetic pathways.

TriLink, the modified nucleic acids experts, offers each of these oxidized forms of mC for inclusion into oligonucleotides to help advance your research of the methylation/demethylation pathway. We also offer many of these modified bases as deoxynucleoside triphosphates, if you wish to investigate alternative potential functions for these important naturally occurring modified bases.

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