Abstract
Abstract
Ten-eleven translocation (TET) dioxygenases initiate active DNA demethylation by sequentially oxidizing 5-methylcytosine (5mC) to 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxylcytosine. We used quantitative LC-MS/MS to investigate how local DNA sequence and substrate concentration influence human TET2-mediated oxidation of CpG (mCG) and non-CpG (mCA) methylcytosines. TET2 exhibited modest flanking-sequence preferences for mCG but substantially stronger sequence dependence for mCA. Systematic analysis identified CCGmCGGC and CTGmCACC as the most efficiently oxidized mCG and mCA substrates, respectively, among sequences examined. Sequence-dependent differences were greater for higher oxidation products than for 5-hydroxymethylcytosine, and relative preferences were retained in a distinct DNA backbone and with full-length TET2. Strikingly, substrate concentration altered these preferences: substrates preferred at higher concentrations became less favored as concentration decreased, shifting relative preference toward AT-rich substrates in both mCG and mCA contexts. Preferred and less-preferred substrates exhibited similar equilibrium binding affinities, indicating that DNA-binding affinity alone does not explain catalytic discrimination. Preferred mCA contexts also overlapped with CAC and CAG contexts associated with DNMT3A and DNMT3B activity. Together, these findings establish local DNA sequence and substrate concentration as interconnected determinants of TET2-mediated methylcytosine oxidation.