fC and caC are formed from iterative oxidation
of mC without release
of hmC. (A) Iterative versus sequential oxidation models. Tet2 is
shown in green, heavy substrates as filled circles, and light as open
circles with dotted lines. Tet2 complexed with heavy *mC-containing
DNA can directly proceed to heavy fC and caC by iterative oxidation.
In sequential oxidation, release of heavy hmC would result in mixing
in solution with light hmC, generating predominantly light fC and
caC. (B) 5 μg/mL Tet2 was incubated for 10 min with heavy *mCpG-containing
duplexes mixed in a 3:1 ratio with light CpG-, mCpG-, or hmCpG-containing
duplexes (100 nM total). Shown is the fraction of heavy isotope for
each modification, as analyzed by LC-MS/MS. (C) Under similar conditions,
Tet2 was incubated with varying ratios of *mC-containing DNA to light
hmC-containing DNA. Shown is the heavy fraction of products, compared
to the heavy fraction of substrate. The mean and SD are shown from
triplicate experiments.