Evaluation of N‐Mustard Analogues of S‐Adenosyl‐L‐methionine with Eukaryotic DNA Methyltransferase 1

Abstract

DNA methylation, which requires the universal methyl donor Sadenosyl-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 alkynefunctionalized 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 physio-logically-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 alkynefunctionality appears to directly impact binding to DNMT1.[a] Dr.

Irreversible Inhibition of DNMT3A by an N-Mustard Analog of S-Adenosyl-L-methionine

“…8 To accomplish this, a synthetic 1998 base pair linearized DNA segment containing two CpG recognition sites for DNMT3AC which overlap with BstUI recognition sites was employed to enable facile visualization following agarose gel electrophoresis. 9 Preliminary experiments indicated that endogenous SAM was co-purified in the E. coli expression of DNMT3AC, as control reactions in the absence of added SAM yielded nearly fully protected DNA (data not shown). The amount of co-purified SAM was reduced through multiple rounds of dialysis to ensure that experiments carried out in the presence of 1 reflected modification of the DNA by the analog and not SAM-dependent methylation.…”