scholarly journals Subdividing Repressor Function: DNA Binding Affinity, Selectivity, and Allostery Can Be Altered by Amino Acid Substitution of Nonconserved Residues in a LacI/GalR Homologue†

Biochemistry ◽  
2008 ◽  
Vol 47 (31) ◽  
pp. 8058-8069 ◽  
Author(s):  
Hongli Zhan ◽  
Marc Taraban ◽  
Jill Trewhella ◽  
Liskin Swint-Kruse
Keyword(s):  
Amino Acid ◽  
Dna Binding ◽  
Binding Affinity ◽  
Amino Acid Substitution ◽  
Dna Binding Affinity

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

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Krystyna Ślaska-Kiss ◽  
Nikolett Zsibrita ◽  
Mihály Koncz ◽  
Pál Albert ◽  
Ákos Csábrádi ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Dna Binding ◽  
Binding Affinity ◽  
Dna Methyltransferase ◽  
Restriction Enzymes ◽  
Dna Binding Protein ◽  
E Coli ◽  
Dna Binding Affinity ◽  
Higher Eukaryotes

AbstractTargeted 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.

scholarly journals Quantification of transcription factor-DNA binding affinity in a living cell

2015 ◽  
Vol 44 (7) ◽  
pp. 3045-3058 ◽  
Author(s):  
Sergey Belikov ◽  
Otto G. Berg ◽  
Örjan Wrange
Keyword(s):  
Transcription Factor ◽  
Dna Binding ◽  
Binding Affinity ◽  
Living Cell ◽  
Dna Binding Affinity
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