Mechanism of CpG DNA Methyltransferases M.SssI and Dnmt3a Studied by DNA Containing 2-Aminopurine

Abstract

Murine DNA methyltransferases Dnmt3a-CD and M.SssI from Spiroplasma methylate cytosines at CpG sites. The role of 6-oxo groups of guanines in DNA methylation by these enzymes has been studied using DNA substrates, which contained 2-aminopurine at different positions. Removal of the 6-oxo group of the guanine located adjacent to the target cytosine in the CpG site dramatically reduces the stability of the methyltransferase-DNA complexes and leads to a significant decrease in the methylation. Apparently, O6 of this guanine is involved in the recognition of CpG sites by the enzymes. Cooperative binding of Dnmt3a-CD to 2-aminopurine-containing DNA and the formation of nonproductive enzyme-substrate complexes were observed.

DNA methylation is regulated by both the stability and topology of G-quadruplex

“…We selected M.SssI MTase because it is a unique methyltransferase among prokaryotic enzymes featured in specific methylation of the CpG sequence in DNA 23 and is used as a model for studying eukaryotic CpG methylation. 24 After methylation, the product was refolded in the buffer for HhaI digestion to form duplexes so that G4 formation would not interfere with restriction enzyme digestion. The gel confirms that the entire DNA forms duplexes in HhaI buffer (Fig.…”

“…Although the structure of M.SssI MTase used in this study has not been solved yet, previous studies have shown that the mechanisms of MTase are similar even in eukaryotes and prokaryotes. 24,29 For example, M. Mpel MTase from M. penetrans interacts with both strands of substrate DNA by Phe133, Glu184, Arg228, Arg230, Ser304 and Phe302 with hydrogen bonding (Fig. S14a, ESI †).…”