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

DNMT2酶是高度保守的，存在于从裂殖酵母到人类的各种物种中，这表明DNMT2酶在细胞动态平衡中起着非常重要的作用。然而，大多数DNMT2基因敲除的生物体最初缺乏明显的表型，因此DNMT2‘S的生物学功能尚不清楚。尽管它们的序列和结构与DNA甲基转移酶相似，但2006年发现DNMT2是一种高度活性的RNA甲基转移酶，能够甲基化tRNA-Asp的C38位。在广泛的前期工作的基础上，并与FOR1082的其他小组密切合作，我们计划在这里研究DNMT2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DNMT2的结构和作用机制的理解，特别是关于RNA和可能的DNA底物识别。我们将研究如下问题：DNMT2如何识别其RNA靶标？是否存在在体外具有很强DNA甲基化活性的DNMT2蛋白，或者是否可以产生这样的变异体？DNMT2是否甲基化具有不寻常结构的DNA？主题区域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氨基-tRNA-合成酶识别tRNA-Asp？C38甲基化是否会影响tRNA误充电的速度？C38甲基化是否影响体内的一般翻译过程？DNMT2基因敲除细胞蛋白质组的改变是否与c38甲基化对tRNA充电和/或一般翻译的影响有关？主题区3以DNMT2的分子进化为例研究多功能酶的进化。主要问题是：DNMT2的进化史是什么？我们能否协调并在实验上跟踪其底物专一性从DNA到tRNA的变化？在细菌物种中发现的唯一DNMT2酶的作用是什么？我们能否通过实验影响DNMT2的特异性，并最终从序列中预测其功能？

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?