Publications by authors named "Angie Kirchner"

7 Publications

  • Page 1 of 1

5-Hydroxymethyl-, 5-Formyl- and 5-Carboxydeoxycytidines as Oxidative Lesions and Epigenetic Marks.

Chemistry 2021 Mar 26. Epub 2021 Mar 26.

Ludwigs-Maximilian-Universität München, Butenandtstr. 5-13, 81377, Munich, Germany.

The four non-canonical nucleotides in the human genome 5-methyl-, 5-hydroxymethyl-, 5-formyl- and 5-carboxydeoxycytidine (mdC, hmdC, fdC and cadC) form a second layer of epigenetic information that contributes to the regulation of gene expression. Formation of the oxidized nucleotides hmdC, fdC and cadC requires oxidation of mdC by ten-eleven translocation (Tet) enzymes that require oxygen, Fe(II) and α-ketoglutarate as cosubstrates. Although these oxidized forms of mdC are widespread in mammalian genomes, experimental evidence for their presence in fungi and plants is ambiguous. This vagueness is caused by the fact that these oxidized mdC derivatives are also formed as oxidative lesions, resulting in unclear basal levels that are likely to have no epigenetic function. Here, we report the xdC levels in the fungus Amanita muscaria in comparison to murine embryonic stem cells (mESCs), HEK cells and induced pluripotent stem cells (iPSCs), to obtain information about the basal levels of hmdC, fdC and cadC as DNA lesions in the genome.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/chem.202100551DOI Listing
March 2021

Active turnover of genomic methylcytosine in pluripotent cells.

Nat Chem Biol 2020 12 10;16(12):1411-1419. Epub 2020 Aug 10.

Department of Chemistry, Ludwig Maximilians University Munich and Center for Integrated Protein Science Munich (CIPSM), Munich, Germany.

Epigenetic plasticity underpins cell potency, but the extent to which active turnover of DNA methylation contributes to such plasticity is not known, and the underlying pathways are poorly understood. Here we use metabolic labeling with stable isotopes and mass spectrometry to quantitatively address the global turnover of genomic 5-methyl-2'-deoxycytidine (mdC), 5-hydroxymethyl-2'-deoxycytidine (hmdC) and 5-formyl-2'-deoxycytidine (fdC) across mouse pluripotent cell states. High rates of mdC/hmdC oxidation and fdC turnover characterize a formative-like pluripotent state. In primed pluripotent cells, the global mdC turnover rate is about 3-6% faster than can be explained by passive dilution through DNA synthesis. While this active component is largely dependent on ten-eleven translocation (Tet)-mediated mdC oxidation, we unveil additional oxidation-independent mdC turnover, possibly through DNA repair. This process accelerates upon acquisition of primed pluripotency and returns to low levels in lineage-committed cells. Thus, in pluripotent cells, active mdC turnover involves both mdC oxidation-dependent and oxidation-independent processes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41589-020-0621-yDOI Listing
December 2020

5-Formylcytosine to cytosine conversion by C-C bond cleavage in vivo.

Nat Chem Biol 2018 Jan 27;14(1):72-78. Epub 2017 Nov 27.

Center for Integrated Protein Science Munich CiPSM at the Department of Chemistry, Ludwig-Maximilians-Universität München, Munich, Germany.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/nchembio.2531DOI Listing
January 2018

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

J Am Chem Soc 2017 08 21;139(30):10359-10364. Epub 2017 Jul 21.

Center for Integrated Protein Science at the Department of Chemistry, LMU Munich , Butenandtstrasse 5-13, Munich 81377, Germany.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/jacs.7b04131DOI Listing
August 2017

5-Formylcytosine Could Be a Semipermanent Base in Specific Genome Sites.

Angew Chem Int Ed Engl 2016 09 25;55(39):11797-800. Epub 2016 Aug 25.

Center for Integrated Protein Science at the Department of Chemistry, Ludwig-Maximilians-Universität München, Butenandtstrasse 5-13, 81377, München, Germany.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/anie.201605994DOI Listing
September 2016

An integrated study of the affinities of the Aβ16 peptide for Cu(I) and Cu(II): implications for the catalytic production of reactive oxygen species.

Metallomics 2014 Mar 3;6(3):505-17. Epub 2014 Feb 3.

The Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Victoria 3010, Australia.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1039/c4mt00001cDOI Listing
March 2014

A study of cyanotetrazole oxides and derivatives thereof.

Chem Asian J 2013 Jan 21;8(1):148-59. Epub 2012 Nov 21.

Department of Chemistry, Energetic Materials Research, Ludwig-Maximilian University, Butenandtstr. 5-13 (D), 81377 München, Germany.

In this work we report on the syntheses of energetic salts of cyanotetrazolate-1- and -2-oxides; this offers a unique ability to compare the effects of tetrazole 1- versus 2-oxidation. 5-Cyanotetrazolate-2-oxide can be synthesized by oxidation of the 5-cyanotetrazolate anion with Oxone, while the corresponding 1-oxide was synthesized by the rearrangement of azidoaminofurazan. Both chemical (multinuclear NMR, IR, and Raman spectroscopies, mass spectrometry, etc.) as well as explosive (impact, friction, and static sensitivities) properties are reported for these energetic salts. Calculated explosive performances using the EXPLO5 computer code are also reported. We furthermore detail the chemistry of these two anions, and their ability to form tetrazole-carboxamides, dihydrotetrazines, and tetrazines. The ability to hydrolyze cyanotetrazole oxides to their amides was demonstrated by two copper complexes. Several crystal structures of these species are presented in addition to full chemical characterization. Finally, the unique 1,4,-bis(2-N-oxidotetrazolate)-1,2,4,5-tetrazine anion was characterized as an energetic material as its ammonium salt.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/asia.201200903DOI Listing
January 2013