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Enzymatic Hydroxylation and Excision of Extended 5-Methylcytosine Analogues

Authors
  • Tomkuvienė, Miglė1
  • Ikasalaitė, Diana1
  • Slyvka, Anton2
  • Rukšėnaitė, Audronė1
  • Ravichandran, Mirunalini3
  • Jurkowski, Tomasz P.4
  • Bochtler, Matthias2, 5
  • Klimašauskas, Saulius1
  • 1 Institute of Biotechnology, Life Sciences Center, Vilnius University, Vilnius LT-10257, Lithuania
  • 2 International Institute of Molecular and Cell Biology, 02-109 Warsaw, Poland
  • 3 School of Medicine, University of California, San Francisco, CA 94143, USA
  • 4 School of Biosciences, Cardiff University, CF10 3AX Cardiff, Wales, UK
  • 5 Polish Academy of Sciences, Institute of Biochemistry and Biophysics, 02-106 Warsaw, Poland
Type
Published Article
Journal
Journal of Molecular Biology
Publisher
Elsevier
Publication Date
Nov 20, 2020
Volume
432
Issue
23
Pages
6157–6167
Identifiers
DOI: 10.1016/j.jmb.2020.10.011
PMID: 33065111
PMCID: PMC7763475
Source
PubMed Central
Keywords
Disciplines
  • Communication
License
Unknown

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 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 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.

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