Biochemical characterization and molecular evolution of the Dnmt2 RNA/DNA metyltransferase

Dnmt2 RNA/DNA 甲基转移酶的生化特征和分子进化

• 批准号: 119070130
• 负责人: Professor Dr. Albert Jeltsch
• 金额: --
• 依托单位: Abteilung für Biochemie
• 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 2014-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/119070130?language=en
• 关键词: Biochemical; characterization; molecular; evolution; Dnmt2

Dnmt2 enzymes are strongly conserved and found in species ranging from Schizosaccharomyces pombe to human, suggesting a very important role for cellular homeostasis. However, most of dnmt2 knock-out organism originally lacked an apparent phenotype such that Dnmt2's biological function remained unknown. Despite their sequence and structural similarity to DNA methyltransferases, it was discovered in 2006 that Dnmt2 is a highly active RNA methyltransferase capable of methylating the C38 position of the tRNA-Asp. Based on extensive preliminary work and in close cooperation with the other groups of FOR1082, we plan to investigate here three important features of the mechanism, function and evolution of Dnmt2. Subject area 1 has the aim to improve our understanding of the structure and mechanism of action of Dnmt2 in particular with respect to RNA and possibly DNA substrate recognition. We will study questions like: How does Dnmt2 recognize its RNA targets? Are there Dnmt2 proteins with strong in vitro DNA methylation activity or can such variants be generated? Does Dnmt2 methylate DNA with unusual structures? Subject area 2 aims to investigate the biological function of the tRNA-Asp C38 methylation catalyzed by Dnmt2. This includes experiments which address questions like: Does C38 methylation contribute to the recognition of tRNA-Asp by Aspartyl-tRNA-synthetase? Does C38 methylation influence the rate of mischarging this tRNA? Does C38 methylation influence the general translation process in vivo? Are there changes in the proteome of Dnmt2 knock-out cells that can be correlated with the effect of the C38 methylation on tRNA charging and/or general translation? Subject area 3 studies the molecular evolution of Dnmt2 as an example of the evolution of a multifunctional enzyme. Main questions are: What is the evolutionary history of Dnmt2? Can we reconcile and experimentally follow its change in substrate specificity from DNA to tRNA? What is the role of the only Dnmt2 enzyme found in a bacterial species? Can we experimentally influence the specificity of Dnmt2 and eventually predict function from sequence?

Dnmt 2酶是高度保守的，并且在从粟酒裂殖酵母到人类的物种中发现，这表明细胞内稳态的非常重要的作用。然而，大多数dnmt 2基因敲除的生物体最初缺乏表型，使得Dnmt 2的生物学功能仍然未知。尽管它们的序列和结构与DNA甲基转移酶相似，但在2006年发现Dnmt 2是一种高活性的RNA甲基转移酶，能够甲基化tRNA-Asp的C38位置。基于广泛的前期工作，并与FOR 1082的其他小组密切合作，我们计划在这里研究Dnmt 2的机制，功能和进化的三个重要特征。主题领域1的目的是提高我们对Dnmt 2的结构和作用机制的理解，特别是在RNA和可能的DNA底物识别方面。我们将研究以下问题：Dnmt 2如何识别其RNA靶标？是否存在具有强体外DNA甲基化活性的Dnmt 2蛋白，或者可以产生这样的变体？Dnmt 2会甲基化具有异常结构的DNA吗？主题领域2旨在研究Dnmt 2催化的tRNA-Asp C38甲基化的生物学功能。这包括解决以下问题的实验：C38甲基化是否有助于天冬氨酰-tRNA-合成酶识别tRNA-Asp？C38甲基化会影响这种tRNA的误充电率吗？C38甲基化是否影响体内的一般翻译过程？Dnmt 2基因敲除细胞的蛋白质组是否存在与C38甲基化对tRNA充电和/或一般翻译的影响相关的变化？主题领域3研究Dnmt 2的分子进化，作为多功能酶进化的一个例子。主要问题是：Dnmt 2的进化历史是什么？我们能否调和并通过实验跟踪它从DNA到tRNA的底物特异性变化？在细菌物种中发现的唯一Dnmt 2酶的作用是什么？我们能否通过实验影响Dnmt 2的特异性，并最终从序列中预测功能？