| Title |
Selective chemical tracking of Dnmt1 catalytic activity in live cells / |
| Authors |
Stankevičius, Vaidotas ; Gibas, Povilas ; Masiulionytė, Bernadeta ; Gasiulė, Liepa ; Masevičius, Viktoras ; Klimašauskas, Saulius ; Vilkaitis, Giedrius |
| DOI |
10.1016/j.molcel.2022.02.008 |
| Full Text |
|
| Is Part of |
Molecular cell.. Cambridge, MA : Cell Press. 2022, vol. 82, iss. 5, p. 1053-1065, e1-e8.. ISSN 1097-2765. eISSN 1097-4164 |
| Keywords [eng] |
5-methylcytosine ; DNA methyltransferase ; cofactor selectivity ; electroporation of AdoMet analogs ; embryonic stem cells ; enzyme engineering ; epigenetic regulation ; in cellulo labeling |
| Abstract [eng] |
Enzymatic methylation of cytosine to 5-methylcytosine in DNA is a fundamental epigenetic mechanism involved in mammalian development and disease. DNA methylation is brought about by collective action of three AdoMet-dependent DNA methyltransferases, whose catalytic interactions and temporal interplay are poorly understood. We used structure-guided engineering of the Dnmt1 methyltransferase to enable catalytic transfer of azide tags onto DNA from a synthetic cofactor analog, Ado-6-azide, in vitro. We then CRISPR-edited the Dnmt1 locus in mouse embryonic stem cells to install the engineered codon, which, following pulse internalization of the Ado-6-azide cofactor by electroporation, permitted selective azide tagging of Dnmt1-specific genomic targets in cellulo. The deposited covalent tags were exploited as "click" handles for reading adjoining sequences and precise genomic mapping of the methylation sites. The proposed approach, Dnmt-TOP-seq, enables high-resolution temporal tracking of the Dnmt1 catalysis in mammalian cells, paving the way to selective studies of other methylation pathways in eukaryotic systems. |
| Published |
Cambridge, MA : Cell Press |
| Type |
Journal article |
| Language |
English |
| Publication date |
2022 |
| CC license |
|
