Application of single-enzyme kinetics to investigate the turnover rate, processivity and specificity of DNA methyltransferase 1

应用单酶动力学研究 DNA 甲基转移酶 1 的周转率、持续合成能力和特异性

• 批准号: 403074082
• 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: Priority Programmes
• 财政年份: 2018
• 资助国家: 德国
• 起止时间: 2017-12-31 至 2022-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/403074082?language=en
• 关键词: Application; single; enzyme; kinetics; investigate

DNMT1 is the main enzyme responsible to copy DNA methylation patterns in human cells, which have fundamental roles in development and disease. It is a large multidomain Protein which is regulated by conformational changes and at least two auto-inhibitory domains. We will employ novel approaches, single enzyme kinetics with long hemimethylated DNA substrates and conformationally locked DNMT1 mutants, to solve fundamental mechanistic questions of DNMT1 and relate its biochemical properties with its biological function in the cell. Key questions of this application are to determine the catalytic rate of DNMT1 during processive DNA methylation, the specificity of DNMT1 for hemimethylated CpG sites, the role of interacting proteins and post translational modifications in the regulation of activity and specificity and the influence of conformational changes in these processes. Our preliminary data indicate that this project will provide important new insights into the function and mechanism of this essential DNA modification enzyme. Our project addresses major research questions in the field of molecular epigenetics namely the detailed molecular mechanism of the accurate inheritance of DNA methylation patterns and the process of epigenetic information transfer in general. Moreover, the project addresses a major challenge in molecular enzymology, which is the question how the activity of large multidomain enzymes are allosterically regulated by conformational changes and binding of interacting proteins.

DNMT1是负责复制人类细胞中DNA甲基化模式的主要酶，其在发育和疾病中起着重要作用。它是一种大的多结构域蛋白，受构象变化和至少两个自抑制结构域的调节。我们将采用新的方法，单酶动力学与长半甲基化的DNA底物和构象锁定的DNMT1突变体，解决DNMT1的基本机制问题，并将其生化特性与其在细胞中的生物学功能联系起来。这个应用程序的关键问题是要确定DNMT1的催化速率进行性DNA甲基化过程中，DNMT1的特异性半甲基化的CpG位点，相互作用的蛋白质和翻译后修饰的活性和特异性的调节中的作用，并在这些过程中的构象变化的影响。我们的初步数据表明，该项目将提供重要的新见解的功能和机制，这种必要的DNA修饰酶。我们的项目解决了分子表观遗传学领域的主要研究问题，即DNA甲基化模式准确遗传的详细分子机制和表观遗传信息传递的一般过程。此外，该项目还解决了分子酶学中的一个主要挑战，即大型多结构域酶的活性如何通过相互作用蛋白的构象变化和结合进行变构调节的问题。