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Enzymatic hydroxylation and excision of extended 5-methylcytosine analogues

Tomkuviene, Migle, Ikasalaite, Diana, Slyvka, Anton, Ruksenaite, Audrone, Ravichandran, Mirunalini, Jurkowski, Tomasz P., Bochtler, Matthias and Klimasauskas, Saulius 2020. Enzymatic hydroxylation and excision of extended 5-methylcytosine analogues. Journal of Molecular Biology 432 (23) , pp. 6157-6167. 10.1016/j.jmb.2020.10.011

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Abstract

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 multi-step 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 Csingle bondC 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 sp1 or sp2 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.

Item Type: Article
Date Type: Publication
Status: Published
Schools: Biosciences
Additional Information: This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Publisher: Elsevier
ISSN: 0022-2836
Date of First Compliant Deposit: 7 December 2020
Date of Acceptance: 8 October 2020
Last Modified: 08 Dec 2020 10:45
URI: http://orca.cf.ac.uk/id/eprint/136838

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