DNA Methyltransferases - Role and Function pp 491-509 | Cite as
Engineering and Directed Evolution of DNA Methyltransferases
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.
Keywords
Directed Evolution Gene Library Methylation Site Enzyme Variant Consensus Amino AcidAbbreviations
- MTase
DNA methyltransferase
- Dnmt
Mammalian DNA methyltransferase
- R/M
Restriction/modification
- IPTG
Isopropyl β-D-1-thiogalactopyranoside
- IVC
In vitro compartmentalization
- PCR
Polymerase chain reaction
- MeDIP
Methylated DNA immunoprecipitation
- CpG
5’-C-phosphate-G-3’
- NMR
Nuclear magnetic resonance
- ELISA
Enzyme-linked immunosorbent assay
- DIG
Digoxigenin
- SDS-PAGE
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis
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