Increasing DNA substrate specificity of the EcoDam DNA-(adenine N6)-methyltransferase by site-directed mutagenesis
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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.
Key wordsEcoDam rational design protein engineering
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