Biochemistry and biological function of mammalian Dnmt1

DNA甲基化是哺乳动物的一个基本过程，参与了几种基因调控的情况。到目前为止，在哺乳动物中已经发现了三种活性的DNA甲基转移酶：参与维持甲基化的DNMT1和参与从头DNA甲基化的DNMT3a和3b。DNMT1是一个由1620个氨基酸残基组成的大蛋白。在胚胎发育过程中，完全敲除DNMT1酶是致命的。在体内，DNMT1的数量减少会导致DNA去甲基化。此外，它还能阻止DNA复制并诱导细胞凋亡。如果这些影响是由于DNA去甲基化造成的，目前还不清楚。DNMT1是磷酸化的，在体外可以与其他几种蛋白质相互作用。生化数据表明，它还可能与一种称为甲基化扩散的过程有关，这种过程通常在肿瘤进展过程中被观察到。在这项应用中，我们打算制备带有单一或少数氨基酸交换的DNMT1变体，这些氨基酸交换专门针对蛋白质的特定生化功能，并在体外鉴定突变的酶。随后，将产生在其内源性DNMT1基因中包含相同定义交换的转基因小鼠，以研究DNMT1某些特性的体内功能。按照这种结合的方法，我们试图调查a)Dnmt1催化活性的丧失是否导致完全敲除的胚胎死亡，b)Dnmt1与增殖细胞核抗原蛋白的相互作用是否在体内起作用，c)体外观察到的Dnmt1的磷酸化是否与体内相关，以及d)体外观察到的Dnmt1的变构激活对未修饰DNA甲基化是否有作用，并可能导致甲基化的扩散。到目前为止，这种结合了定点突变和基因打靶的实验还没有用任何DNA甲基转移酶进行。最后，我们试图通过制备小鼠基因敲除株来研究另一种DNA甲基转移酶DNMT2的生物学作用，并对转基因动物进行了几代的仔细研究。我们预计，这些实验的结果将大大加深我们对DNA甲基化和表观遗传基因控制的了解。

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.