Functional characterization of the Dnmt2 homolog DnmA in Dictyostelium

在Forschergruppe(FOR)的框架内，该项目将继续深入了解网柄苔藓中甲基转移酶DnmA的生物化学和功能。我们将研究新发现的底物RNA的功能，并继续尝试识别与DnmA相关的蛋白质成分和DNA序列。我们的数据表明，DnmA在细胞周期和发育过程中受到调控。我们将设计实验，阐明DnmA在细胞生命周期中的这些特定时间点的功能。在一些野生分离株中显著较高的表达水平将极大地促进这一方法。DNMT2的一个有趣的特征是它在进化上高度保守，这与缺乏明显的突变表型形成了鲜明对比。更令人惊讶的是，观察到KO细胞在混合培养中似乎比野生型细胞具有生长优势。我们将阐明导致这一意外发现的因素。我们希望定义需要DnmA的条件，从而为其进化保守功能提供指征。针对DnmA和hDnmt2的特异性单链抗体已经获得，并将被优化用于检测内源性蛋白。与前一个资助期一样，将与For的合作伙伴密切合作开展试验。只要有可能，比较研究将与来自不同模式生物的DNMT2同源物并行进行。

In the frame of the Forschergruppe (FOR), this project will continue to obtain insights into the biochemistry and function of the methyltransferase DnmA in Dictyostelium. We will investigate the function of newly identified substrate RNAs and continue attempts to identify protein components as well as DNA sequences that associate with DnmA. Our data suggest regulation of DnmA during the cell cycle and during development. We will design experiments that will elucidate DnmA functions at these specific time points in the life cycle of a cell. The significantly higher expression levels in some wild isolates will substantially facilitate this approach. An intriguing feature of Dnmt2 is its high conservation in evolution which is contrasted by the lack of obvious mutant phenotypes. Even more surprising is the observation that KO cells appear to have a growth advantage over wild type cells in mixed cultures. We will elucidate the factors responsible for this unexpected finding. We hope to define conditions where DnmA is required and thus provide indications for its evolutionary conserved function. Specific single chain antibodies against DnmA and hDnmt2 have been obtained and will be optimised to detect the endogenous proteins. As in the previous funding period, experiments will be carried out in close cooperation with the partners in the FOR. Wherever possible, comparative studies will be done in parallel with Dnmt2 homologues from the different model organisms.