RNA-Substrate Sepcificity of Dnmt2

DNMT2是真核细胞DNA甲基转移酶家族中的一员，但它能有效催化tRNA甲基化。目前，不同tRNA38位的核糖体-5-甲基胞苷(ribo-M5C)的作用尚不清楚，而Dnmt1和Dnmt3形成的脱氧-M5C残基被认为是经典的表观遗传标记。虽然两种类型的核酸MTase的核酸修饰催化机制相似，但它们的功能似乎有很大的差异，但DNA甲基化可能仍然是DNMT2活性的一部分。在这种背景下，该项目研究了DNMT2对核酸底物的识别，作为一种特征生化特征，在DNMT2与其他MTase的比较中提供了信息。到目前为止，我们的分析表明，除了反密码子结构域中包含七个核苷酸的共识基序外，大多数tRNA结构都是必需的。精细的分析将通过基于DNMT2底物与辅因子类似物的催化烷基化的组合方法来执行。转移的烷基含有末端的炔基，它可以通过点击化学连接到生物素上，以物理分离DNMT2底物。然后进行深度测序或扩增以进行新的选择。在另一项研究中，我们发现在甲基化位点及其周围含有多达10个脱氧核苷酸的tRNAs是DNMT2的有效底物。我们将研究DNA含量最高的杂交底物的甲基化活性，以评估DNMT2与其他DNMT酶和其他MTase的生化相似性程度。进一步的具有不同RNA和DNA含量的核酸杂交，其中两种类型的核酸要么是共价连接的，要么是通过退火法组装的，它们将被开发成一个由DNMT2引导的体外甲基化系统。同时，还将开展胞苷甲基化的LC-MS分析和选择性化学修饰。

Dnmt2 is a well aligned member of the eukaryotic family of DNA methyltransferases, but it efficiently catalyzes tRNA methylation. The role of the resulting ribo‐5‐methylcytidine (ribo‐m5C) at position 38 of different tRNAs is poorly understood at present, while the desoxy‐m5C residues formed by Dnmt1 and Dnmt3 are thought to be classical epigenetic marks. While the catalytic mechanism of nucleic acid modification between the two types of nucleic acid MTases is similar, their functions seem to strongly diverge, yet DNA methylation may still be part of the Dnmt2 activity. In this backdrop, the project addresses the nucleic acid substrate recognition by Dnmt2 as a characteristic biochemical feature that is informative in the comparison of Dnmt2 with other MTases. Our analysis so far shows that most of the tRNA structure is required in addition to a consensus motif comprising seven nucleotides in the anticodon domain. A refined analysis will be performed by a combinatorial approach based on catalytic alkylation of Dnmt2 substrates with a cofactor analogue. The transferred alkylgroup contains a terminal alkyne, which can be conjugated to biotin by click chemistry for physical separation of Dnmt2 substrates. This is followed by deep sequencing or by amplification for a renewed selection. In another line of investigation, we have found that tRNAs containing stretches of up to ten deoxynucleotides at and around the methylation site are efficient substrates of Dnmt2. We will investigate methylation activity of hybrid substrates with maximized DNA content, to assess the degree of biochemical similarity of Dnmt2 to other Dnmt enzymes and further MTases. Further nucleic acid hybrids with varying contents of RNA and DNA, in which both types of nucleic acids are either covalently linked, or assembled by annealing, will be developed into an in vitro system for guided methylation by Dnmt2. Accompanying analytics of cytidine methylation by LC‐MS and selective chemical modification will be developed.