Lowering DNA binding affinity of SssI DNA methyltransferase does not enhance the specificity of targeted DNA methylation in E. coli

Abstract Targeted DNA methylation is a technique that aims to methylate cytosines in selected genomic loci. In the most widely used approach a CG-specific DNA methyltransferase (MTase) is fused to a sequence specific DNA binding protein, which binds in the vicinity of the targeted CG site(s). Althou...

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Autores principales:	Krystyna Ślaska-Kiss, Nikolett Zsibrita, Mihály Koncz, Pál Albert, Ákos Csábrádi, Sarolta Szentes, Antal Kiss
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2021
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/e93f3b9e48ad4b848a4c095544d60062
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spelling	oai:doaj.org-article:e93f3b9e48ad4b848a4c095544d600622021-12-02T16:24:52ZLowering DNA binding affinity of SssI DNA methyltransferase does not enhance the specificity of targeted DNA methylation in E. coli10.1038/s41598-021-94528-32045-2322https://doaj.org/article/e93f3b9e48ad4b848a4c095544d600622021-07-01T00:00:00Zhttps://doi.org/10.1038/s41598-021-94528-3https://doaj.org/toc/2045-2322Abstract Targeted DNA methylation is a technique that aims to methylate cytosines in selected genomic loci. In the most widely used approach a CG-specific DNA methyltransferase (MTase) is fused to a sequence specific DNA binding protein, which binds in the vicinity of the targeted CG site(s). Although the technique has high potential for studying the role of DNA methylation in higher eukaryotes, its usefulness is hampered by insufficient methylation specificity. One of the approaches proposed to suppress methylation at unwanted sites is to use MTase variants with reduced DNA binding affinity. In this work we investigated how methylation specificity of chimeric MTases containing variants of the CG-specific prokaryotic MTase M.SssI fused to zinc finger or dCas9 targeting domains is influenced by mutations affecting catalytic activity and/or DNA binding affinity of the MTase domain. Specificity of targeted DNA methylation was assayed in E. coli harboring a plasmid with the target site. Digestions of the isolated plasmids with methylation sensitive restriction enzymes revealed that specificity of targeted DNA methylation was dependent on the activity but not on the DNA binding affinity of the MTase. These results have implications for the design of strategies of targeted DNA methylation.Krystyna Ślaska-KissNikolett ZsibritaMihály KonczPál AlbertÁkos CsábrádiSarolta SzentesAntal KissNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 11, Iss 1, Pp 1-11 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Krystyna Ślaska-Kiss Nikolett Zsibrita Mihály Koncz Pál Albert Ákos Csábrádi Sarolta Szentes Antal Kiss Lowering DNA binding affinity of SssI DNA methyltransferase does not enhance the specificity of targeted DNA methylation in E. coli
description	Abstract Targeted DNA methylation is a technique that aims to methylate cytosines in selected genomic loci. In the most widely used approach a CG-specific DNA methyltransferase (MTase) is fused to a sequence specific DNA binding protein, which binds in the vicinity of the targeted CG site(s). Although the technique has high potential for studying the role of DNA methylation in higher eukaryotes, its usefulness is hampered by insufficient methylation specificity. One of the approaches proposed to suppress methylation at unwanted sites is to use MTase variants with reduced DNA binding affinity. In this work we investigated how methylation specificity of chimeric MTases containing variants of the CG-specific prokaryotic MTase M.SssI fused to zinc finger or dCas9 targeting domains is influenced by mutations affecting catalytic activity and/or DNA binding affinity of the MTase domain. Specificity of targeted DNA methylation was assayed in E. coli harboring a plasmid with the target site. Digestions of the isolated plasmids with methylation sensitive restriction enzymes revealed that specificity of targeted DNA methylation was dependent on the activity but not on the DNA binding affinity of the MTase. These results have implications for the design of strategies of targeted DNA methylation.
format	article
author	Krystyna Ślaska-Kiss Nikolett Zsibrita Mihály Koncz Pál Albert Ákos Csábrádi Sarolta Szentes Antal Kiss
author_facet	Krystyna Ślaska-Kiss Nikolett Zsibrita Mihály Koncz Pál Albert Ákos Csábrádi Sarolta Szentes Antal Kiss
author_sort	Krystyna Ślaska-Kiss
title	Lowering DNA binding affinity of SssI DNA methyltransferase does not enhance the specificity of targeted DNA methylation in E. coli
title_short	Lowering DNA binding affinity of SssI DNA methyltransferase does not enhance the specificity of targeted DNA methylation in E. coli
title_full	Lowering DNA binding affinity of SssI DNA methyltransferase does not enhance the specificity of targeted DNA methylation in E. coli
title_fullStr	Lowering DNA binding affinity of SssI DNA methyltransferase does not enhance the specificity of targeted DNA methylation in E. coli
title_full_unstemmed	Lowering DNA binding affinity of SssI DNA methyltransferase does not enhance the specificity of targeted DNA methylation in E. coli
title_sort	lowering dna binding affinity of sssi dna methyltransferase does not enhance the specificity of targeted dna methylation in e. coli
publisher	Nature Portfolio
publishDate	2021
url	https://doaj.org/article/e93f3b9e48ad4b848a4c095544d60062
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Lowering DNA binding affinity of SssI DNA methyltransferase does not enhance the specificity of targeted DNA methylation in E. coli

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