Distributive Processing by the Iron(II)/α-Ketoglutarate-Dependent Catalytic Domains of the TET Enzymes Is Consistent with Epigenetic Roles for Oxidized 5-Methylcytosine Bases

The ten-eleven translocation (TET) proteins catalyze oxidation of 5-methylcytosine (C-5m) residues in nucleic acids to 5-hydroxymethylcytosine (C-5hm), 5-formylcytosine (C-5f), and 5-carboxycytosine (C-5ca). These nucleotide bases have been implicated as intermediates on the path to active demethylation, but recent reports have suggested that they might have specific regulatory roles in their own right. In this study, we present kinetic evidence showing that the catalytic domains (CDs) of TET2 and TETI from mouse and their homologue from Naegleria gruberi, the full-length protein NgTET1, are distributive in both chemical and physical senses, as they carry out successive oxidations of a single C-5m and multiple C-5m residues along a polymethylated DNA substrate. We present data showing that the enzyme neither retains C-5hm/C-5f intermediates of preceding oxidations nor slides along a DNA substrate (without releasing it) to process an adjacent C-5m residue. These findings contradict a recent report by Crawford et al. (j Am. Chem. Soc. 2016, 138, 730) claiming that oxidation of C-5m by CD of mouse TET2 is chemically processive (iterative). We further elaborate that this distributive mechanism is maintained for TETs in two evolutionarily distant homologues and posit that this mode of function allows the introduction of smC forms as epigenetic markers along the DNA.