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修饰是胞嘧啶甲基化，主要是在CpG中 二核苷酸。正常DNA甲基化模式的破坏已知在几种疾病中发挥作用。 DNA甲基转移酶1(DNMT1)主要负责维持DNA甲基化模式 通过多轮细胞分裂。DNMT1是一种具有C末端催化作用的多结构域蛋白 甲基转移酶结构域和一个大的N末端调控区。复制焦点定位 序列(RFTS)结构域位于N-末端调节区，是DNMT1活性的关键调节因子 活着。RFTS结构域与DNA结合部位结合，阻止与DNA的结合。此外， RFTS结构域参与了几种蛋白质-蛋白质相互作用，这些相互作用有助于定位和激活DNMT1 催化作用。最近，RFTS结构域的突变被鉴定为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修改的DNA甲基化模式， 低甲基化和定点高甲基化。导致这一现象的分子机制发生了变化 甲基化模式仍然不清楚，对这些因素的生化后果知之甚少。 突变。在这项建议中，我们试图了解这些氨基酸序列的变化是如何影响 DNMT1的结构和功能。对于特定目标1，熔化温度和荧光各向异性将 用于检测由疾病相关突变引起的蛋白质稳定性和动力学的变化。 对于特定的目的2，将在突变的酶中评估RFTS介导的自抑制。两者的DNA结合 将检测野生型和突变型酶的亲和力和DNA甲基化活性，以确定 突变解除了正常的自我抑制。对于特定目标3，突变对关键分子间的影响 我们将研究交互作用。已知RFTS结构域与uhrf1(泛素样物，包含PHD)结合 和环指域蛋白1)和修饰组蛋白H3尾巴。将使用等温滴定量热法 研究RFTS突变对这些调节蛋白-蛋白质相互作用的影响。我们的预赛 研究表明，与疾病相关的突变G589A和V590F降低了蛋白质的热稳定性，而 也提高了DNA结合亲和力和催化活性，表明至少部分缓解了正常的RFTS- 在这些突变的酶中介导性的自我抑制。总体而言，这些研究代表了一种极好的培训 为本科生提供机会。本科生将从事这项研究的所有方面，并获得 具有设计实验、收集和分析数据以及解释结果的实践经验。我们预计 我们的生化研究为疾病相关突变的后果提供了关键的见解 最终将有助于我们理解受影响的疾病形成的分子机制 个人。

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.