A directed evolution design of a GCG-specific DNA hemimethylase

Abstract

DNA cytosine-5 methyltransferases (C5-MTases) are valuable models to study sequence-specific modification of DNA and are becoming increasingly important tools for biotechnology. Here we describe a structure-guided rational protein design combined with random mutagenesis and selection to change the specificity of the HhaI C5-MTase from GCGC to GCG. The specificity change was brought about by a five-residue deletion and introduction of two arginine residues within and nearby one of the target recognizing loops. DNA protection assays, bisulfite sequencing and enzyme kinetics showed that the best selected variant is comparable to wild-type M.HhaI in terms of sequence fidelity and methylation efficiency, and supersedes the parent enzyme in transalkylation of DNA using synthetic cofactor analogs. The designed C5-MTase can be used to produce hemimethylated CpG sites in DNA, which are valuable substrates for studies of mammalian maintenance MTases.

Engineering the DNA cytosine-5 methyltransferase reaction for sequence-specific labeling of DNA

“…The observed rates were still lower as compared to those achieved with the natural cofactor (9), although in theory the S N 2 alkylation rates with propargyl and allyl compounds should be similar or higher than the methylation rates (21). Directed evolution of M.HhaI for altered sequence specificity produced variants in which Tyr254 is replaced with serine, and which also showed an enhanced transalkylation activity with certain extended cofactor analogs (17). To guide our engineering effort, we built a structure-based model of M.HhaI–DNA–(cofactor 2 ) complex (Figure 2A and B).…”

“…Cofactor 2 was obtained as a diasteromeric mixture of R,S- and S,S- isomers starting from butyn-2-ol-1 (16). Cofactor 3 was synthesized starting from pentyn-2-ol-1 and purified to an over 85% chiral purity of the enzymatically active S,S -isomer (17). For analog 4, the alkylation was performed with N -BOC-protected 6-(4-aminobutanamido)hex-2-yn-1-yl 4-nitrobenzenesulfonate, which was obtained in three steps from 5-chloropentyne-1 following previously described procedures (10), and the S,S -diastereomer was isolated by reversed-phase HPLC.…”

Approaches to Enzyme and Substrate Design of the Murine Dnmt3a DNA Methyltransferase

“…So far, this approach had been successfully applied to prokaryotic DNA MTases, which in general have high catalytic activity. [19][20][21][22][23] Here we have attempted to produce Dnmt3a-C variants with improved catalytic activity by directed evolution. In addition, we investigated the flankingsequence preferences of Dnmt3a-C for methylation of DNA.…”

Die Methyltransferase‐gesteuerte Markierung von Biomolekülen und ihre Anwendungen

“…Dies könnte zurückzuführen sein auf sperrige Reste an den AdoMet‐Analoga, die eine gute sterische Einpassung verhindern, oder auf die Störung der erforderlichen Bindungswechselwirkung mit der Bindungstasche für AdoMet. Bei diesen Szenarien könnte es sich als nützlich erweisen, die AdoMet‐Bindungstasche so zu gestalten, dass eine günstigere Wechselwirkung mit den AdoMet‐Analoga erreicht wird, beispielsweise durch gerichtete Evolution der MTase …”