Investigating the Impact of Disease-Associated Mutations on DNA Methyltransferase 1 Function

项目摘要 在人类中，最常见的表观遗传DNA修饰是胞嘧啶的甲基化，主要在CpG中 二核苷酸。已知正常DNA甲基化模式的破坏在几种疾病中起作用。 DNA甲基转移酶1（DNMT 1）主要负责维持DNA甲基化模式 通过多轮细胞分裂DNMT 1是一种具有C末端催化活性的多结构域蛋白质 甲基转移酶结构域和大的N-末端调节区。复制焦点定位 在N-末端调节区发现的序列（RFTS）结构域是DNMT 1活性的关键调节因子， vivo. RFTS结构域与DNA结合位点结合并阻止与DNA结合。此外该 RFTS结构域参与几种蛋白质-蛋白质相互作用，其用于定位和激活DNMT 1， 催化作用最近，RFTS结构域的突变已被确定，导致两种不同的成人发病 神经退行性疾病受影响的个体表现出改变的DNA甲基化模式， 低甲基化和位点特异性高甲基化。导致这种改变的分子机制 甲基化模式仍然不清楚，并且对这些甲基化的生物化学后果知之甚少。 突变在这项提案中，我们试图了解这些氨基酸序列的变化如何影响 DNMT 1的结构和功能。对于具体目标1，解链温度和荧光各向异性将 用于检查由疾病相关突变引起的蛋白质稳定性和动力学的变化。 对于具体目标2，将在突变酶中评估RFTS介导的自身抑制。DNA结合 将在野生型和突变型酶中检查亲和力和DNA甲基化活性，以确定 突变解除正常的自身抑制。对于具体目标3，突变对关键分子间 互动将被审查。已知RFTS结构域结合UHRF 1（泛素样，含有PHD 和RING指结构域蛋白1）和修饰的组蛋白H3尾。将使用等温滴定量热法 研究RFTS突变对这些调节蛋白-蛋白相互作用的影响。我们的初步 研究表明，疾病相关突变G589 A和V590 F降低了蛋白质的热稳定性， 也增加DNA结合亲和力和催化活性，表明正常RFTS至少部分缓解， 介导的自身抑制。总的来说，这些研究代表了一个很好的培训 本科生的机会。本科生将从事这项研究的各个方面，并获得 实践经验设计实验，收集和分析数据，并解释结果。我们预计 我们的生物化学研究，以产生关键的见解的后果，疾病相关的突变， 将最终帮助我们理解疾病形成的分子机制， 个体

Project Summary In humans, the most common epigenetic DNA modification is methylation of cytosines, predominantly in CpG dinucleotides. Disruption of the normal DNA methylation pattern is known to play a role in several diseases. DNA methyltransferase 1 (DNMT1) is primarily responsible for maintenance of the DNA methylation pattern through multiple rounds of cell division. DNMT1 is a multidomain protein with a C-terminal catalytic methyltransferase domain and a large N-terminal regulatory region. The Replication Focus Targeting Sequence (RFTS) domain, found in the N-terminal regulatory region, is a key regulator of DNMT1 activity in vivo. The RFTS domain binds to the DNA binding site and prevents association with DNA. In addition, the RFTS domain is involved in several protein-protein interactions that serve to localize and activate DNMT1 for catalysis. Recently, mutations in the RFTS domain have been identified that result in two different adult onset neurodegenerative disorders. Affected individuals exhibit modified DNA methylation patterns with global hypomethylation and site-specific hypermethylation. The molecular mechanisms that lead to this altered methylation pattern are still unclear and little is known about the biochemical consequences of these mutations. In this proposal, we seek to understand how these changes in amino acid sequence are impacting the structure and function of DNMT1. For specific aim 1, melting temperatures and fluorescence anisotropy will be used to examine changes in protein stability and dynamics induced by the disease-associated mutations. For specific aim 2, RFTS-mediated autoinhibition will be assessed in the mutant enzymes. Both DNA binding affinity and DNA methylation activity will be examined in wild-type and mutant enzymes to determine if the mutations relieve normal autoinhibition. For specific aim 3, the impact of the mutations on key intermolecular interactions will be examined. The RFTS domain is known to bind to UHRF1 (ubiquitin-like, containing PHD and RING finger domains protein 1) and modified histone H3 tails. Isothermal titration calorimetry will be used to investigate the impact of RFTS mutations on these regulatory protein-protein interactions. Our preliminary work shows that disease-associated mutations G589A and V590F reduce thermal stability of the protein while also increasing DNA binding affinity and catalytic activity, indicating at least partial relief of normal RFTS- mediated autoinhibition in these mutant enzymes. Collectively, these studies represent an excellent training opportunity for undergraduate students. Undergraduates will engage in all aspects of this research and gain hands-on experience designing experiments, collecting and analyzing data, and interpreting results. We expect our biochemical studies to yield key insights into the consequences of the disease-associated mutations that will ultimately aid in our understanding of the molecular mechanisms of disease formation in affected individuals.