Increasing DNA substrate specificity of the ecodam DNA--methyltransferasen by site-directed mutagenesis

Increasing DNA substrate specificity of the ecodam DNA--methyltransferasen by site-directed mutagenesis

DNA methylation catalyzed by DNA methyltransferases (MTases) is widespread in prokaryotes. In an attempt to find EcoDam variants with enhanced preference for hemimethylated DNA, the L122, P134, and V133 residues were replaced with other amino acids using site directed mutagenesis, and the catalytic...

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Veröffentlicht in:Biochemistry (Moscow) 2014-11, Vol.79 (11), p.1262
Hauptverfasser: Elsawy, H, Chahar, S
Format: Artikel
Sprache:eng
Schlagworte:
Analysis
Chemical properties
Control
Design and construction
DNA-ligand interactions
Evaluation
Genetic aspects
Methods
Methylation
Methyltransferases
Mutagenesis
Protein engineering
Structure
Substrates (Biochemistry)
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Zusammenfassung:DNA methylation catalyzed by DNA methyltransferases (MTases) is widespread in prokaryotes. In an attempt to find EcoDam variants with enhanced preference for hemimethylated DNA, the L122, P134, and V133 residues were replaced with other amino acids using site directed mutagenesis, and the catalytic activity of all variants on unmethylated and hemimethylated substrates was studied. Our results showed that, in addition to L122A, the L122S and L122A/V133L EcoDam variants were able to sense the methylation status of the 5'-GATC-3' double-stranded target recognition site and methylated only hemimethylated DNA. DOI: 10.1134/S0006297914110145 Key words: EcoDam, rational design, protein engineering
ISSN:0006-2979
DOI:10.1134/S0006297914110145