1 research outputs found
5‑Formyl- and 5‑Carboxydeoxycytidines Do Not Cause Accumulation of Harmful Repair Intermediates in Stem Cells
5-Formyl-dC
(fdC) and 5-carboxy-dC (cadC) are newly discovered
bases in the mammalian genome that are supposed to be substrates for
base excision repair (BER) in the framework of active demethylation.
The bases are recognized by the monofunctional thymine DNA glycosylase
(Tdg), which cleaves the glycosidic bond of the bases to give potentially
harmful abasic sites (AP-sites). Because of the turnover of fdC and
cadC during cell state transitions, it is an open question to what
extent such harmful AP-sites may accumulate during these processes.
Here, we report the development of a new reagent that in combination
with mass spectrometry (MS) allows us to quantify the levels of AP-sites.
This combination also allowed the quantification of β-elimination
(βE) products, which are repair intermediates of bifunctional
DNA glycosylases. In combination with feeding of isotopically labeled
nucleosides, we were able to trace the intermediates back to their
original nucleobases. We show that, while the steady-state levels
of fdC and cadC are substantially increased in Tdg-deficient cells,
those of both AP- and βE-sites are unaltered. The levels of
the detected BER intermediates are 1 and 2 orders of magnitude lower
than those of cadC and fdC, respectively. Thus, neither the presence
of fdC nor that of cadC in stem cells leads to the accumulation of
harmful AP- and βE-site intermediates