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

Dnmt2是一种高度保守的酶，存在于从裂糖菌到人类的各种物种中，表明其在细胞稳态中起着非常重要的作用。然而，大多数dnmt2敲除生物最初缺乏明显的表型，因此dnmt2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Dnmt2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和与FOR1082其他小组的密切合作，我们计划在此研究Dnmt2的机制、功能和演化的三个重要特征。主题领域1的目的是提高我们对Dnmt2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。我们将研究这样的问题：Dnmt2如何识别它的RNA靶标？是否存在Dnmt2蛋白具有较强的体外DNA甲基化活性或可以产生这样的变体？Dnmt2会甲基化结构不寻常的DNA吗？课题2旨在研究Dnmt2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酸- trna合成酶对tRNA-Asp的识别？C38甲基化是否影响该tRNA的错充率？体内C38甲基化是否影响一般的翻译过程？Dnmt2敲除细胞的蛋白质组中是否存在与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Dnmt2的分子进化，作为多功能酶进化的一个例子。主要问题是：Dnmt2的进化史是怎样的？我们可以调和和实验跟踪底物特异性从DNA到tRNA的变化吗？在细菌物种中发现的唯一的Dnmt2酶的作用是什么？我们可以通过实验影响Dnmt2的特异性并最终从序列预测功能吗？

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?