期刊
JOURNAL OF MOLECULAR BIOLOGY
卷 432, 期 23, 页码 6157-6167出版社
ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD
DOI: 10.1016/j.jmb.2020.10.011
关键词
DNA cytosine-5 methylation; TET dioxygenase; DNA glycosylase; epigenetic regulation; radical intermediate
资金
- European Research Council [ERC-2016-AdG/742654]
- Foundation for Polish Science/EU Regional Development Fund [POIR.04.04.00-00-5D81/1700, START 79.2020]
- Polish National Academic Exchange agency (NAWA) [PPI/APM/2018/1/00034]
- Deutsche Forschungsgemeinschaft [JU 2773-2]
Methylation of cytosine to 5-methylcytosine (mC) is a prevalent reversible epigenetic mark in vertebrates established by DNA methyltransferases (MTases); the methylation mark can be actively erased via a multistep demethylation mechanism involving oxidation by Ten-eleven translocation (TET) enzyme family dioxygenases, excision of the latter oxidation products by thymine DNA (TDG) or Nei-like 1 (NEIL1) glycosylases followed by base excision repair to restore the unmodified state. Here we probed the activity of the mouse TET1 (mTET1) and Naegleria gruberi-TET (nTET) oxygenases with DNA substrates containing extended derivatives of the 5-methylcytosine carrying linear carbon chains and adjacent unsaturated C-C bonds. We found that the nTET and mTET1 enzymes were active on modified mC residues in single-stranded and double-stranded DNA in vitro, while the extent of the reactions diminished with the size of the extended group. Iterative rounds of nTET hydroxylations of ssDNA proceeded with high stereo specificity and included not only the natural alpha position but also the adjoining carbon atom in the extended side chain. The regioselectivity of hydroxylation was broken when the reactive carbon was adjoined with an sp(1) or sp(2) system. We also found that NEIL1 but not TDG was active with bulky TET-oxidation products. These findings provide important insights into the mechanism of these biologically important enzymatic reactions. (C) 2020 The Author(s). Published by Elsevier Ltd.
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