RNA-Substrate Sepcificity of Dnmt2

Dnmt2 的 RNA 底物特异性

• 批准号: 119070036
• 负责人: Professor Dr. Mark Helm
• 金额: --
• 依托单位: Institut für Pharmazeutische und Biomedizinische Wissenschaften (IPBW)
• 依托单位国家: 德国
• 项目类别: Research Units
• 财政年份: 2009
• 资助国家: 德国
• 起止时间: 2008-12-31 至 2015-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/119070036?language=en
• 关键词: RNA; Substrate; Sepcificity; Dnmt2

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.

Dnmt2是真核生物DNA甲基转移酶家族的一个很好的成员，但它有效地催化tRNA甲基化。目前，人们对不同trna 38位的核糖- 5‐甲基胞苷（核糖- m5C）的作用知之甚少，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。虽然两种核酸mtase对核酸修饰的催化机制相似，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。在此背景下，该项目将Dnmt2的核酸底物识别作为一种典型的生化特征，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，除了在反密码子结构域中包含7个核苷酸的共识基序外，大多数tRNA结构是必需的。精细化的分析将通过基于催化烷基化Dnmt2底物与辅助因子类似物的组合方法进行。转移的烷基含有一个末端炔，可以通过点击化学与生物素结合，用于Dnmt2底物的物理分离。随后进行深度测序或扩增以重新选择。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十个脱氧核苷酸延伸的trna是Dnmt2的有效底物。我们将研究具有最大DNA含量的杂交底物的甲基化活性，以评估Dnmt2与其他Dnmt酶和其他mt酶的生化相似性程度。进一步具有不同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，要么是通过退火组装的，将被开发成Dnmt2引导甲基化的体外系统。随后将开发LC - MS和选择性化学修饰的胞苷甲基化分析。