Rational Manipulation of DNA Methylation by Using Isotopically Reinforced Cytosine |
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| Authors: | Clayton B Woodcock Egor A Ulashchik Dr Nikolai E Poopeiko Dr Vadim V Shmanai Prof Norbert O Reich Dr Mikhail S Shchepinov |
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| Affiliation: | 1. Department of Chemistry and Biochemistry, University of California Santa Barbara, Santa Barbara, CA, USA;2. Institute of Physical Organic Chemistry, National Academy of Science of Belarus, Minsk, Belarus;3. Retrotope, Inc., Los Altos, CA, USA |
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| Abstract: | The human DNA methyltransferase 3A (DNMT 3A) is responsible for de novo epigenetic regulation, which is essential for mammalian viability and implicated in diverse diseases. All DNA cytosine C5 methyltransferases follow a broadly conserved catalytic mechanism. We investigated whether C5 β‐elimination contributes to the rate‐limiting step in catalysis by DNMT3A and the bacterial M.HhaI by using deuterium substitutions of C5 and C6 hydrogens. This substitution caused a 1.59–1.83 fold change in the rate of catalysis, thus suggesting that β‐elimination is partly rate‐limiting for both enzymes. We used a multisite substrate to explore the consequences of slowing β‐elimination during multiple cycles of catalysis. Processive catalysis was slower for both enzymes, and deuterium substitution resulted in DNMT 3A dissociating from its substrate. The decrease in DNA methylation rate by DNMT 3A provides the basis of our ongoing efforts to alter cellular DNA methylation levels without the toxicity of currently used methods. |
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| Keywords: | DNA epigenetics isotope effects methyltransferases processivity |
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