Abstract
AbstractDNA methylation, which requires the universal methyl donor S‐adenosyl‐L‐methionine (SAM), plays a pivotal role in eukaryotic gene regulation and when dysregulated, can result in severe alterations in cellular function. An emerging approach to further understand DNA methylation utilizes azide‐ and alkyne‐functionalized N‐mustard SAM analogues as biochemical tools to probe sites of DNA methylation. While the successful utility of these substituted analogues has been demonstrated with prokaryotic DNA methyltransferases, their utility with physiologically‐relevant eukaryotic DNA methyltransferase 1 (DNMT1) is examined for the first time here. A fluorescence‐based magnetic bead assay was validated in initial experiments to measure the extent of DNA modification by the N‐mustard analogues using Spiroplasma methylase, M.SssI, a prokaryotic model of DNMT1. Subsequent analysis with DNMT1 revealed limited utility of the analogues, as added azide‐ and alkyne‐functionality appears to directly impact binding to DNMT1.
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