Chapter title |
Engineering and Directed Evolution of DNA Methyltransferases.
|
---|---|
Chapter number | 18 |
Book title |
DNA Methyltransferases - Role and Function
|
Published in |
Advances in experimental medicine and biology, November 2016
|
DOI | 10.1007/978-3-319-43624-1_18 |
Pubmed ID | |
Book ISBNs |
978-3-31-943622-7, 978-3-31-943624-1
|
Authors |
Paola Laurino, Liat Rockah-Shmuel, Dan S. Tawfik |
Editors |
Albert Jeltsch, Renata Z. Jurkowska |
Abstract |
DNA methyltransferases (MTases) constitute an attractive target for protein engineering, thus opening the road to new ways of manipulating DNA in a unique and selective manner. Here, we review various aspects of MTase engineering, both methodological and conceptual, and also discuss future directions and challenges. Bacterial MTases that are part of restriction/modification (R/M) systems offer a convenient way for the selection of large gene libraries, both in vivo and in vitro. We review these selection methods, their strengths and weaknesses, and also the prospects for new selection approaches that will enable the directed evolution of mammalian DNA methyltransferases (Dnmts). We explore various properties of MTases that may be subject to engineering. These include engineering for higher stability and soluble expression (MTases, including bacterial ones, are prone to misfolding), engineering of the DNA target specificity, and engineering for the usage of S-adenosyl-L-methionine (AdoMet) analogs. Directed evolution of bacterial MTases also offers insights into how these enzymes readily evolve in nature, thus yielding MTases with a huge spectrum of DNA target specificities. Engineering for alternative cofactors, on the other hand, enables modification of DNA with various groups other than methyl and thus can be employed to map and redirect DNA epigenetic modifications. |
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