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

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.

Function and disruption of DNA Methyltransferase 3a cooperative DNA binding and nucleoprotein filament formation

“…This result indicated that Dnmt3a-C bound to the DNA without specificity for CpG sites. The slightly better binding to the non-CG substrate might be due to the flanking sequence, which has a strong influence on the DNA interaction and DNA methylation of Dnmt3a that is well documented [see (31) and references therein]. It is also possible that the energy needed for flipping of the target cytosine may reduce the binding to CpG site substrates [see (36) and references therein for examples of prokaryotic DNA methyltransferase studied in this respect].…”

“…All the kinetic reactions were carried out at least three times. DNA methylation was also measured using a 520-bp DNA fragment containing 40 CpG sites was amplified from phage λ DNA using a biotinylated PCR primer as described (31). …”

Allosteric control of mammalian DNA methyltransferases – a new regulatory paradigm

“…In addition to their preference for the methylation of CpG sites, both DNMT3A and DNMT3B are very sensitive to the sequences flanking their target sites. This is illustrated by the finding that CpG sites embedded in certain flanking sequences cannot be methylated by DNMT3A at all ( 139 ). It has been shown that purine bases are preferred at the 5′ end of the CpG sites, whereas pyrimidines are favored at their 3′ end ( 139 – 141 ).…”

Target specificity of mammalian DNA methylation and demethylation machinery

“…Although in comparison to prokaryotic methyltransferases DNMT3A and DNMT3B do not seem to have strong sequence specificity beyond CpG sites, both enzymes are very sensitive to the sequences flanking their target sites. DNMT3A prefers purine bases at the 5' end of the CpG sites, whereas pyrimidines are favoured at their 3' end [80][81][82] . These so called flanking sequence preferences of the methyltransferases might have strong impact on the generation of methylation patterns, as CpG sites embedded unfavourable flanking sequence context cannot be methylated by DNMT3A at all 81 .…”

“…DNMT3A prefers purine bases at the 5' end of the CpG sites, whereas pyrimidines are favoured at their 3' end [80][81][82] . These so called flanking sequence preferences of the methyltransferases might have strong impact on the generation of methylation patterns, as CpG sites embedded unfavourable flanking sequence context cannot be methylated by DNMT3A at all 81 . Interestingly, experimental flanking sequence preferences of DNMT3A and DNMT3B correlate with the statistical data on the methylation level of CpG sites found in the human genome 80,83 , suggesting that the inherent sequence preferences of de novo enzymes contribute to the selection of genomic regions that undergo methylation.…”