3,N4-Etheno-5-methylcytosine blocks TET1-3 oxidation but is repaired by ALKBH2, 3 and FTO

Abstract

5-Methyldeoxycytidine (5mC) is a major epigenetic marker that regulates cellular functions in mammals. Endogenous lipid peroxidation can convert 5mC into 3,N4-etheno-5-methylcytosine (ε5mC). ε5mC is structurally similar to the mutagenic analog 3,N4-ethenocytosine (εC), which is repaired by AlkB family enzymes in the direct reversal repair (DRR) pathway and excised by DNA glycosylases in the base excision repair (BER) pathway. However, the repair of ε5mC has not been reported. Here, we examined the activities against ε5mC by DRR and BER enzymes and TET1-3, enzymes that modify the 5-methyl group in 5mC. We found that the etheno modification of 5mC blocks oxidation by TET1-3. Conversely, three human homologs in the AlkB family, ALKBH2, 3 and FTO were able to repair ε5mC to 5mC, which was subsequently modified by TET1 to 5-hydroxymethylcytosine. We also demonstrated that ALKBH2 likely repairs ε5mC in MEF cells. Another homolog, ALKBH5, could not repair ε5mC. Also, ε5mC is not a substrate for BER glycosylases SMUG1, AAG, or TDG. These findings indicate DRR committed by ALKBH2, 3 and FTO could reduce the detrimental effects of ε5mC in genetics and epigenetics and may work together with TET enzymes to modulate epigenetic regulations.