Biochemical characterisation of TET DNA hydroxylases

Abstract

Methylation of DNA in CpG dinucleotide plays an important role in mammalian development. The recent discovery of TET enzymes showed that DNA demethylation can occur through stepwise oxidation of 5-methylcytosine (5mC) to 5-hyrdoxymethylcytosine (5hmC), 5-formylcytosine (5fC) and finally to 5-carboxylcytosine (5-caC) followed by the removal of the higher oxidised bases by Thymine DNA glycosylase (TDG) and base excision repair mechanism. Genetic studies revealed that the TET enzymes are involved in numerous biological processes such as transcriptional regulation, hematopoietic stem cell differentiation, embryonic, primodial germ cells (PGCs) development and are commonly misregulated in cancer. While the biological functions of TET enzymes have been studied extensively, very little is known about their biochemical properties. In this body of work, the biochemistry of TET enzymes was investigated in detail with the focus on their catalytic and kinetic behaviour, which would allow us to understand the molecular mechanisms of TET enzymes. First of all, an in vitro system including a novel plate assay to quantify the oxidation products catalysed by TET enzymes, was established. As a proof of principle, several analogues of α-Ketoglutarate, the intermediates of citric acid cycle (oncometabolites) were tested. Moreover, the effect of divalent metal ions was tested both in vitro and in vivo and it was demonstrated that the addition of nickel ions to mammalian cells decreased the level of 5hmC through inhibition of TET enzymes by displacing the Fe2+ from the catalytic centre. Furthermore, using detailed biochemical studies, it was demonstrated that ascorbic acid (AscA) modulates the activity of TET enzymes through efficient recycling of Fe2+, which challenges the existing view of AscA as a bound cofactor of TET enzymes. Finally, using biochemical approach followed by next generation sequencing and bioinformatics analysis, the catalytic behaviour of TET enzymes on single molecule level was elucidated. Using linear double stranded DNA containing multiple 5hmC-substrates in different flanking sequence context, it was shown that mammalian TET enzymes oxidize 5hmC substrates in both CG and non-CG context. Importantly, both mammalian TET enzymes and Naegleria gruberi Tet1 like dioxygenase (nTet) showed a strong and distinct flanking sequence preference. In addition, it was shown that TET enzymes (both mammalian and nTet) might catalyse the substrates on DNA in distributive manner.