5-Formyl- and 5-Carboxydeoxycytidines Do Not Cause Accumulation of Harmful Repair Intermediates in Stem Cells

Article abstract of DOI:10.1021/jacs.7b04131

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.

Full text of DOI:10.1021/jacs.7b04131

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Article
5-Formyl- and 5-carboxydeoxycytidines do not cause
accumulation of harmful repair intermediates in stem cells
René Rahimoff, Olesea Kosmatchev, Angie Kirchner, Toni Pfaffeneder,
Fabio Spada, Victor Brantl, Markus Müller, and Thomas Carell
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.7b04131 • Publication Date (Web): 17 Jul 2017
Downloaded from http://pubs.acs.org on July 20, 2017
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Scheme 1: Synthesis of 1 (red) in a light (1a) and heavy (1b) form and of the light and heavy internal
standards 9a/b and 10a/b (blue).
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Figure 1: Mechanism of the base excision repair process. A) Chemistry of BER with formation of APꢀ and βEꢀ
sites. Blue pathway: Monofunctional glycosylases act in concert with Ape1 and Pol β. Red pathway: Biꢀ
functional glycosylases. B) Depiction of reagent 1 and of the reaction products that are formed when 1
reacts with APꢀ and βEꢀsites.
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Figure 2: A) Fragmentation pattern of 9a/b and 10a/b, which give defined daughter ions after loss of N or
an aryl radical. B) Reaction of reagent 1 with a defined APꢀsite created inside a DNA duplex. Quantification
of the reaction product 9a after the treatment of the oligodeoxynucleotide with 1a. C) UHPLꢀchromatogram
of the canonical bases plus the AP (9a)ꢀ and βEꢀsite (10a) reaction products formed with reagent 1a.
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Figure 3: Quantification of BER intermediates and isotope tracing studies. A) Quantitative data of global
levels of APꢀ and βEꢀsites from mESCs. Inhibition and knockdown of Ape1 yields an increased amount of APꢀ
sites. B) Feeding of mESC cultures with labelled nucleosides results in the formation of riboseꢀlabelled APꢀ
and βEꢀsite products 9* and 10* which are five mass units heavier than unlabelled products.
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Figure 4: Quantification of labelled APꢀ and βEꢀintermediates of the isotope tracing studies and quantification
of the fdC and cadC content in Tdg wt and KO cells. n.d., not detectable. (i) levels were over the LOD, but
under LLOQ.
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