Increasing DNA Substrate Specificity of the EcoDam DNA-(adenine N⁶)-methyltransferase by Site-Directed Mutagenesis

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H. Elsawy^1,2* and S. Chahar³

¹Chemistry Department, Faculty of Science, Tanta University, Tanta, Egypt; E-mail: Hany.mostafa@science.tanta.edu.eg

²Current address: College of Science, King Faisal University, Al-Ahsaa, P.B. Box 380, Hufof 31982, Saudi Arabia

³Department of Genetics, Rutgers, The State University of New Jersey, New Brunswick, New Jersey, USA; E-mail: chahars@googlemail.com

^* To whom correspondence should be addressed.

Received February 4, 2014; Revision received June 26, 2014

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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.

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KEY WORDS: EcoDam, rational design, protein engineering

DOI: 10.1134/S0006297914110145

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