Approaches to enzyme and substrate design of the murine Dnmt3a DNA methyltransferase

Jurkowska, Renata Z. ORCID: https://orcid.org/0000-0002-4507-2222, Siddique, Abu Nasar, Jurkowski, Tomasz P. ORCID: https://orcid.org/0000-0002-2012-0240 and Jeltsch, Albert 2011. Approaches to enzyme and substrate design of the murine Dnmt3a DNA methyltransferase. ChemBioChem 12 (10) , 1589—1594. 10.1002/cbic.201000673

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Abstract

Dnmt3a‐C, the catalytic domain of the Dnmt3a DNA‐(cytosine‐C5)‐methyltransferase, is active in an isolated form but, like the full‐length Dnmt3a, shows only weak DNA methylation activity. To improve this activity by directed evolution, we set up a selection system in which Dnmt3a‐C methylated its own expression plasmid in E. coli, and protected it from cleavage by methylation‐sensitive restriction enzymes. However, despite screening about 400 clones that were selected in three rounds from a random mutagenesis library of 60 000 clones, we were not able to isolate a variant with improved activity, most likely because of a background of uncleaved plasmids and plasmids that had lost the restriction sites. To improve the catalytic activity of Dnmt3a‐C by optimization of the sequence of the DNA substrate, we analyzed its flanking‐sequence preference in detail by bisulfite DNA‐methylation analysis and sequencing of individual clones. Based on the enrichment and depletion of certain bases in the positions flanking >1300 methylated CpG sites, we were able to define a sequence‐preference profile for Dnmt3a‐C from the −6 to the +6 position of the flanking sequence. This revealed preferences for T over a purine at position −2, A over G at −1, a pyrimidine at +1, and A and T over G at +3. We designed one “good” substrate optimized for methylation and one “bad” substrate designed not to be efficiently methylated, and showed that the optimized substrate is methylated >20 times more rapidly at its central CpG site. The optimized Dnmt3a‐C substrate can be applied in enzymatic high‐throughput assays with Dnmt3a‐C (e.g., for inhibitor screening), because the increased activity provides an improved dynamic range and better signal/noise ratio.

Item Type:	Article
Date Type:	Publication
Status:	Published
Schools:	Biosciences
Publisher:	Wiley
ISSN:	1439-4227
Last Modified:	22 Dec 2024 15:52
URI:	https://orca.cardiff.ac.uk/id/eprint/118976

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