Tet enzymes oxidize 5-methyl-deoxycytidine (mdC) to 5-hydroxymethyl-dC (hmdC), 5-formyl-dC (fdC) and 5-carboxy-dC (cadC) in DNA. It was proposed that fdC and cadC deformylate and decarboxylate, respectively, to dC over the course of an active demethylation process. This would re-install canonical dC bases at previously methylated sites. However, whether such direct C-C bond cleavage reactions at fdC and cadC occur in vivo remains an unanswered question. Here we report the incorporation of synthetic isotope- and (R)-2'-fluorine-labeled dC and fdC derivatives into the genome of cultured mammalian cells. Following the fate of these probe molecules using UHPLC-MS/MS provided quantitative data about the formed reaction products. The data show that the labeled fdC probe is efficiently converted into the corresponding labeled dC, most likely after its incorporation into the genome. Therefore, we conclude that fdC undergoes C-C bond cleavage in stem cells, leading to the direct re-installation of unmodified dC.

A new fluorescent probe Aβ16wwa based upon the Aβ16 peptide has been developed with two orders of magnitude greater fluorescence intensity for sensitive detection of interactions with Cu(II). In combination with the Cu(I) probe Ferene S, it is confirmed that the Aβ16 peptide binds either Cu(I) or Cu(II) with comparable affinities at pH 7.4 (log K = -10.4; log K = -10.0). It follows from this property that the Cu-Aβ16 complex is a robust if slow catalyst for the aerial oxidation of ascorbate with H2O2 as primary product (initial rate, ∼0.63 min(-1) for Cu-Aβ16 versus >2.5 min(-1) for Cuaq(2+)). An integrated study of variants of this peptide identifies the major ligands and binding modes involved in its copper complexes in solution. The dependence of K upon pH is consistent with a two-coordinate Cu(I) site in which dynamic processes exchange Cu(I) between the three available pairs of imidazole sidechains provided by His6, His13 and His14. The N-terminal amine is not involved in Cu(I) binding but is a key ligand for Cu(II). Acetylation of the N-terminus alters the redox thermodynamic gradient for the Cu centre and suppresses its catalytic activity considerably. The data indicate the presence of dynamic processes that exchange Cu(II) between the three His ligands and backbone amide at physiological pH. His6 is identified as a key ligand for catalysis as its presence minimises the pre-organisation energy required for interchange of the two copper redox sites. These new thermodynamic data strengthen structural interpretations for the Cu-Aβ complexes and provide valuable insights into the molecular mechanism by which copper chemistry may induce oxidative stress in Alzheimer's disease.

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.

5-Formyl-2'-deoxycytosine (fdC) is a recently discovered epigenetic base in the genome of stem cells, with yet unknown functions. Sequencing data show that the base is enriched in CpG islands of promoters and hence likely involved in the regulation of transcription during cellular differentiation. fdC is known to be recognized and excised by the enzyme thymine-DNA-glycosylase (Tdg). As such, fdC is believed to function as an intermediate during active demethylation. In order to understand the function of the new epigenetic base fdC, it is important to analyze its formation and removal at defined genomic sites. Here, we report a new method that combines sequence-specific chemical derivatization of fdC with droplet digital PCR that enables such analysis. We show initial data, indicating that the repair protein Tdg removes only 50 % of the fdCs at a given genomic site, arguing that fdC is a semipermanent base.