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Biochemistry and biological function of mammalian Dnmt1

哺乳动物Dnmt1的生物化学和生物学功能

基本信息

批准号：
5370240
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：
2002
资助国家：
德国
起止时间：
2001-12-31 至 2008-12-31
项目状态：
已结题

来源：
https://gepris.dfg.de/gepris/projekt/5370240?language=en
关键词：
Biochemistry biological function mammalian Dnmt1

项目摘要

DNA methylation is an essential process in mammals involved in several instances of gene regulation. So far, three active DNA methyltransferases have been found in mammals: Dnmt1 involved in maintenance methylation; Dnmt3a and 3b involved in de novo DNA methylation. Dnmt1 is a large protein comprising 1620 amino acid residues. Complete knock-out of the Dnmt1 enzyme is lethal during embryogenesis. Reduction of the amount of Dnmt1 causes demethylation of the DNA in vivo. Furthermore, it leads to an arrest of DNA replication and induces apoptosis. If these effects are due to demethylation of the DNA is not known. Dnmt1 is phosphorylated and interacts with several other proteins in vitro. Biochemical data suggests that it might also be implicated in a process called spreading of methylation, which is often observed in the course of tumor progression. In this application we intend to prepare Dnmt1 variants carrying single or few amino acid exchanges that specifically address defined biochemical functions of the protein and characterize the mutant enzymes in vitro. Subsequently transgenic mice will be generated which contain the same defined exchanges in their endogenous dnmt1 gene to investigate the in vivo function of certain properties of Dnmt1. Following this combined approach, we attempt to investigate a) if the loss of catalytic activity of Dnmt1 is responsible for embryonic lethality of the full knock-out, b) if the interaction of Dnmt1 with the PCNA protein has a role in vivo, c) if phosphorylation of Dnmt1 that has been observed in vitro is of relevance in vivo and d) if an allosteric activation of Dnmt1 for methylation of unmodified DNA that has been observed in vitro and might cause spreading of methylation has a function in vivo. Such experiments which combine site-directed mutagenesis and gene targeting, so far, have not been carried out with any DNA methyltransferase. Finally, we attempt to investigate the biological role of an additional DNA methyltransferase, Dnmt2, whose function so far is not known by preparing a mouse knock-out strain and carefully investigate the transgenic animals over several generations. We expect that the results of these experiments will considerably further our knowledge on DNA methylation and epigenetic gene control.

DNA甲基化是哺乳动物中一个重要的过程，参与多种基因调控。迄今为止，在哺乳动物中发现了三种活跃的DNA甲基转移酶：Dnmt 1参与维持甲基化; Dnmt 3a和3b参与DNA从头甲基化。Dnmt 1是一种由1620个氨基酸残基组成的大蛋白。Dnmt 1酶的完全敲除在胚胎发生期间是致命的。Dnmt 1的量的减少导致体内DNA的去甲基化。此外，它导致DNA复制停滞并诱导细胞凋亡。如果这些影响是由于DNA的去甲基化是未知的。DNMT1在体外被磷酸化并与其他几种蛋白质相互作用。生化数据表明，它也可能涉及一个称为甲基化扩散的过程，这在肿瘤进展过程中经常观察到。在本申请中，我们打算制备携带单个或很少的氨基酸交换的Dnmt 1变体，所述氨基酸交换特异性地解决蛋白质的限定的生化功能并在体外表征突变酶。随后将产生在其内源性dnmt 1基因中含有相同定义的交换的转基因小鼠，以研究Dnmt 1的某些性质的体内功能。根据这种组合方法，我们试图研究a）Dnmt 1的催化活性的丧失是否是完全敲除的胚胎致死性的原因，B）Dnmt 1与PCNA蛋白的相互作用是否在体内起作用，c）如果在体外观察到的Dnmt 1磷酸化在体内具有相关性，以及如果Dnmt 1对未修饰DNA的甲基化的变构激活在体外观察到并可能导致甲基化的扩散在体内具有功能。迄今为止，结合联合收割机和基因靶向的这些实验还没有用任何DNA甲基转移酶进行。最后，我们试图调查的生物学作用的额外的DNA甲基转移酶，Dnmt 2，其功能迄今为止还不知道通过制备小鼠基因敲除株，并仔细研究了几代转基因动物。我们希望这些实验的结果将大大提高我们对DNA甲基化和表观遗传基因控制的认识。