Mechanism and regulation of the Dnmt1 DNA methyltransferase

Dnmt 1是一种大的多结构域酶，其在DNA复制后特异性地甲基化半甲基化CpG位点，从而通过细胞分裂复制现有的DNA甲基化模式，这是涉及几种生物过程的关键表观遗传事件。在我们的提案中，我们想解决的问题是，Dnmt 1的体外活性不足以解释细胞中DNA复制后DNA再甲基化的速度。我们实验室的初步数据表明，Dnmt 1的体外速率被低估了，因为该酶以不同的构象存在。新的结构数据确实表明，Dnmt 1可以采用不同的自抑制构象，其中其他结构域阻止DNA进入催化结构域。我们计划进行单酶动力学，并确定DNA结合后，Dnmt 1在其最活跃的构象的DNA甲基化活性。此外，我们的目标是测量Dnmt 1在细胞中沿着DNA的可能的直接移动（与体外DNA上的随机移动相反）是否可以进一步加速DNA甲基化。我们建议的第二部分的出发点是，Dnmt 1的体外特异性显然不足以确保在没有额外调控的情况下正确复制甲基化模式，因为该酶也对未甲基化的CpG位点显示出活性。当没有正确的半甲基化CpG底物位点可用时，这在S期之外尤其关键。因此，我们的目的是研究是否发生Dnmt 1的细胞周期偶联调节。为此，我们计划确定Dnmt 1的翻译后修饰，并研究其细胞周期连接。在磷酸化和赖氨酸甲基化的情况下，我们将进一步研究它们是否影响酶的活性或特异性或其与其他蛋白质的相互作用。我们还将研究这些修饰是否影响Dnmt 1的亚细胞和亚核定位。我们相信，我们的项目将有助于理解DNA甲基化模式的维持，这是分子表观遗传学的一个基本过程。

Dnmt1 is a large multidomain enzyme which specifically methylates hemimethylated CpG sites after DNA replication and thereby copies exiting DNA methylation patterns through cell divisions which is a key epigenetic event involved in several biological processes. In our proposal, we like to address the question that the in vitro activity of Dnmt1 is not sufficient to explain the speed of remethylation of DNA after DNA replication in cells. Preliminary data from our lab suggest that the in vitro rate of Dnmt1 is underestimated, because the enzyme exists in different conformations. Novel structural data indeed show that Dnmt1 can adopt different autoinhibitory conformations, in which other domains block access of the DNA to the catalytic domain. We plan to carry out single enzyme kinetics and determine the DNA methylation activity of Dnmt1 in its most active conformation after DNA has been bound. In addition, we aim to measure if a possible direct movement of Dnmt1 along DNA in cells (as opposed to the random walk on DNA in vitro) could further speed up DNA methylation. The starting point of the second part of our proposal is that the in vitro specificity of Dnmt1 is obviously not sufficient to ensure correct copying of methylation patterns without additional regulation, because the enzyme also shows activity towards unmethylated CpG sites. This is particularly critical outside of S-phase, when no correct hemimethylated CpG substrate sites are available. Therefore, we aim to study if cell cycle coupled regulation of Dnmt1 occurs. To this end, we plan to identify post-translational modifications of Dnmt1 and study their cell cycle connection. In the case of phosphorylations and lysine methylations we will further study, whether they influence the enzymes activity or specificity or its interaction with other protein. We will also investigate if the modifications affect the sub-cellular and sub-nuclear localisation of Dnmt1. We are convinced that our project will contribute important pieces to the understanding of the maintenance of DNA methylation patterns a fundamental process in molecular epigenetics.