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In Vitro Directed Evolution of a Restriction Endonuclease with More Stringent Specificity
Published on: March 25, 2020
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Engineering and Directed Evolution of DNA Methyltransferases.
Paola Laurino1, Liat Rockah-Shmuel1, Dan S Tawfik2
1Department of Biological Chemistry, Weizmann Institute of Science, Rehovot, 76100, Israel.
Advances in Experimental Medicine and Biology
|November 10, 2016
Summary
Protein engineering of DNA methyltransferases (MTases) allows novel DNA manipulation. This review covers MTase engineering methods, challenges, and future directions for directed evolution of mammalian DNA methyltransferases (Dnmts).
Area of Science:
- Biochemistry
- Molecular Biology
- Protein Engineering
Background:
- DNA methyltransferases (MTases) are crucial enzymes for DNA modification.
- MTases are attractive targets for protein engineering due to their role in DNA manipulation.
- Bacterial MTases within restriction/modification systems are valuable for gene library selection.
Purpose of the Study:
- To review methodological and conceptual aspects of MTase engineering.
- To discuss future directions and challenges in MTase engineering.
- To explore prospects for directed evolution of mammalian DNA methyltransferases (Dnmts).
Main Methods:
- Review of existing selection methods (in vivo and in vitro) for MTase engineering.
- Exploration of engineering strategies for MTase stability, specificity, and cofactor usage.
- Analysis of directed evolution approaches for bacterial and mammalian MTases.
Main Results:
- MTase engineering can enhance stability, solubility, and alter DNA target specificity.
- Engineering enables the use of S-adenosyl-L-methionine (AdoMet) analogs and alternative cofactors for novel DNA modifications.
- Directed evolution of bacterial MTases provides insights into natural enzyme evolution and specificity.
Conclusions:
- Protein engineering offers unique and selective DNA manipulation capabilities.
- Directed evolution holds promise for developing novel mammalian DNA methyltransferases (Dnmts).
- Engineered MTases can map and redirect DNA epigenetic modifications beyond methylation.
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