Approaches to Enzyme and Substrate Design of the Murine Dnmt3a DNA Methyltransferase
Approaches to Enzyme and Substrate Design of the Murine Dnmt3a DNA Methyltransferase
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DOI：
10.1002/cbic.201000673
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发表时间：
2011-07-04
期刊：
影响因子：
3.2
通讯作者：
Jeltsch, Albert
中科院分区：
文献类型：
--
作者：
Jurkowska, Renata Z.;Siddique, Abu Nasar;Jeltsch, Albert
Dnmt3a- c是Dnmt3a DNA-（胞嘧啶- c5）-甲基转移酶的催化结构域，在分离形式下具有活性，但与全长Dnmt3a一样，仅表现出较弱的DNA甲基化活性。为了通过定向进化提高这一活性，我们建立了Dnmt3a-C在大肠杆菌中甲基化自身表达质粒的选择系统，并通过甲基化敏感的限制性内切酶保护其不被切割。然而，尽管从60000个随机突变文库中分三轮筛选了大约400个克隆，但我们仍未能分离出活性提高的变体，这很可能是由于未切割质粒和质粒失去了限制性位点的背景。为了通过优化DNA底物序列来提高Dnmt3a-C的催化活性，我们通过亚硫酸盐DNA甲基化分析和单个克隆测序详细分析了其侧翼序列偏好。基于bbbb1300甲基化CpG位点两侧某些碱基的富集和缺失，我们能够定义Dnmt3a-C在侧翼序列-6到+6位置的序列偏好谱。这揭示了T比-2位置的嘌呤更受欢迎，a比-1位置的G更受欢迎，+1位置的嘧啶更受欢迎，以及+3位置的a和T更受欢迎。我们设计了一个优化甲基化的“好”底物和一个不被有效甲基化的“坏”底物，结果表明，优化后的底物在其中心CpG位点的甲基化速度提高了20倍。优化后的Dnmt3a-C底物可以应用于Dnmt3a-C的酶促高通量分析（例如抑制剂筛选），因为增加的活性提供了更好的动态范围和更好的信噪比。
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 60000 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.