Changing the recognition specificity of a DNA-methyltransferase by in vitro evolution

Area of Science:

• Molecular Biology
• Enzymology
• Protein Engineering

Background:

• DNA methyltransferases are crucial enzymes that modify DNA bases.
• The SinI methyltransferase specifically recognizes and methylates the GG(A)/(T)CC sequence.
• Understanding enzyme specificity is key for developing novel molecular tools.

Purpose of the Study:

• To engineer the SinI DNA-methyltransferase for relaxed recognition specificity.
• To identify the key amino acid substitutions responsible for altered DNA sequence recognition.
• To characterize the biochemical properties of the engineered enzyme.

Main Methods:

• In vitro mutagenesis and DNA shuffling of the SinI methyltransferase gene.
• Selection for enzymes with relaxed GGNCC recognition specificity.
• Site-directed mutagenesis to pinpoint critical amino acid substitutions.
• Enzyme purification and kinetic characterization (kcat/Km).

Main Results:

• An in vitro evolution experiment yielded a mutant SinI methyltransferase with five amino acid substitutions.
• Two specific substitutions (L214S and Y229H) located between conserved motifs VI and VII were primarily responsible for the relaxed specificity.
• The mutant enzyme exhibited reduced activity on the wild-type GG(A)/(T)CC site but significantly increased activity on the GG(G)/(C)CC site.
• Substitutions in the N-terminal region had a weaker effect on specificity.

Conclusions:

• Amino acid residues outside the previously assumed 'variable region' are critical for SinI methyltransferase sequence specificity.
• Targeted engineering of DNA methyltransferases can successfully alter their recognition patterns.
• The characterized mutant enzyme offers a modified tool for DNA methylation studies and applications.