RNA-Substrate Sepcificity of Dnmt2

Dnmt 2是真核生物DNA甲基转移酶家族的一个成员，但它有效地催化tRNA甲基化。目前对不同tRNA的38位核糖-5-甲基胞苷（ribo-m5 C）的作用了解甚少，而Dnmt 1和Dnmt 3形成的脱氧-m5 C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之间的核酸修饰的催化机制是相似的，但它们的功能似乎强烈不同，然而DNA甲基化可能仍然是Dnmt 2活性的一部分。在此背景下，该项目将Dnmt 2的核酸底物识别作为一种特征性生物化学特征，在Dnmt 2与其他MT酶的比较中提供信息。到目前为止，我们的分析表明，除了在反密码子结构域中包含7个核苷酸的共有基序之外，还需要大部分tRNA结构。将通过基于Dnmt 2底物与辅因子类似物的催化烷基化的组合方法进行精细分析。转移的烷基含有末端炔，其可以通过点击化学与生物素缀合以物理分离Dnmt 2底物。随后进行深度测序或扩增以进行重新选择。在另一条研究路线中，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tRNA是Dnmt 2的有效底物。我们将研究具有最大化DNA含量的杂合底物的甲基化活性，以评估Dnmt 2与其他Dnmt酶和其他MT酶的生化相似性程度。具有不同RNA和DNA含量的进一步核酸杂交体，其中两种类型的核酸共价连接或通过退火组装，将被开发成用于通过Dnmt 2指导甲基化的体外系统。将开发通过LC-MS和选择性化学修饰的胞苷甲基化的伴随分析。

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.