Abstract
Cytosine methylation in CpG dinucleotides is an epigenetic DNA modification dynamically established and maintained by DNA methyltransferases and demethylases. Molecular mechanisms of active DNA demethylation began to surface only recently with the discovery of the 5-methylcytosine (5mC)-directed hydroxylase and base excision activities of ten–eleven translocation (TET) proteins and thymine DNA glycosylase (TDG). This implicated a pathway operating through oxidation of 5mC by TET proteins, which generates substrates for TDG-dependent base excision repair (BER) that then replaces 5mC with C. Yet, direct evidence for a productive coupling of TET with BER has never been presented. Here we show that TET1 and TDG physically interact to oxidize and excise 5mC, and proof by biochemical reconstitution that the TET–TDG–BER system is capable of productive DNA demethylation. We show that the mechanism assures a sequential demethylation of symmetrically methylated CpGs, thereby avoiding DNA double-strand break formation but contributing to the mutability of methylated CpGs.
Highlights
Cytosine methylation in CpG dinucleotides is an epigenetic DNA modification dynamically established and maintained by DNA methyltransferases and demethylases
An engagement of the core base excision repair (BER) system implies that the AP-site is first incised by an AP endonuclease, which generates a DNA single-strand break that engages, through activation of poly (ADP-ribose) polymerase 1, the X-ray repair cross-complementing protein 1 (XRCC1), DNA ligase 3 (LIG3) and DNA polymerase b (POLb) for DNA gap filling with an unmethylated C and ligation[30]
To address the mode of cooperation between ten–eleven translocation (TET) and thymine DNA glycosylase (TDG), we investigated their physical interaction, first by co-expression and affinity purification of a full-length carboxy-terminally 6His-tagged TET1 (TET1–His6) with a C-terminally glutathione S-transferase (GST)-tagged TDG (TDG–GST)
Summary
Cytosine methylation in CpG dinucleotides is an epigenetic DNA modification dynamically established and maintained by DNA methyltransferases and demethylases. An engagement of the core BER system implies that the AP-site is first incised by an AP endonuclease (that is, APE1), which generates a DNA single-strand break that engages, through activation of poly (ADP-ribose) polymerase 1, the X-ray repair cross-complementing protein 1 (XRCC1), DNA ligase 3 (LIG3) and DNA polymerase b (POLb) for DNA gap filling with an unmethylated C and ligation[30] This mechanism is plausible and widely accepted, there is little evidence supporting a direct link between TET and BER; a productive action of TET with the BER system on a 5mC substrate has not been shown, nor have the basic mechanistic features of such a process been addressed. The data proof full functionality of a TET1–TDG–BER-based DNA demethylation system on hemi- and fully methylated DNA, and show that the molecular transactions involved are coordinated in a manner avoiding DNA fragmentation but creating a risk for mutation if deamination and demethylation events coincide within a CpG
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