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

The gene coding for the SinI DNA-methyltransferase, a modification enzyme able to recognize and methylate the internal cytosine of the GGA/TCC sequence, was subjected to in vitro mutagenesis, DNA-shuffling and a strong selection for relaxed GGNCC recognition specificity. As a result of this in vitro...

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Bibliographic Details
Published in:Nucleic acids research 2004-01, Vol.32 (13), p.3898-3903
Main Authors: Tímár, Edit, Groma, Gergely, Kiss, Antal, Venetianer, Pál
Format: Article
Language:English
Subjects:
Amino Acid Substitution
Base Sequence
Directed Molecular Evolution
DNA-Cytosine Methylases - chemistry
DNA-Cytosine Methylases - genetics
DNA-Cytosine Methylases - metabolism
Mutagenesis
Recombination, Genetic
Substrate Specificity
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Summary:The gene coding for the SinI DNA-methyltransferase, a modification enzyme able to recognize and methylate the internal cytosine of the GGA/TCC sequence, was subjected to in vitro mutagenesis, DNA-shuffling and a strong selection for relaxed GGNCC recognition specificity. As a result of this in vitro evolution experiment, a mutant gene with the required phenotype was selected. The mutant SinI methyltransferase carried five amino acid substitutions. None of these was found in the ‘variable region’ that were thought to be responsible for sequence specificity. Three were located near the N-terminal end, preceding the first conserved structural motif of the enzyme; two were found between conserved motifs VI and VII. A clone engineered to carry out only the latter two replacements (L214S and Y229H) displays relaxed recognition specificity similar to that of the parental mutant, whereas the clone carrying only the N-terminal replacements showed a much weaker change in recognition specificity. The enzyme with two internal mutations was purified and characterized. Its catalytic activity (kcat/Km) was ∼5-fold lower towards GGA/TCC and 20-fold higher towards GGG/CCC than that of the wild-type enzyme.
ISSN:0305-1048
1362-4962
1362-4962
DOI:10.1093/nar/gkh724