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

研究疾病相关突变对 DNA 甲基转移酶 1 功能的影响

• 批准号: 10291710
• 负责人: Rebecca Switzer
• 金额: $ 20.26万
• 依托单位: BUCKNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-08-01 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10291710
• 关键词: Aberrant DNA Methylation; Active Sites; Adult; Affect; Affinity; Amino Acid Sequence; Binding; Binding Proteins; Binding Sites; Biochemical; Biological Assay; Biomedical Research; C-terminal; Calorimetry; Catalysis; Catalytic Domain; Cell Culture System; Cell division; Cell physiology; Cells; Coupled; CpG dinucleotide; Cytosine; DNA; DNA Binding; DNA Maintenance; DNA Methylation; DNA Methyltransferase Inhibitor; DNA Modification Methylases; DNA Modification Process; Data; Data Analyses; Development; Disease; Enzyme Kinetics; Enzymes; Epigenetic Process; Exhibits; Exposure to; Family; Fluorescence Anisotropy; Funding Mechanisms; Genes; Goals; Histone H3; Human; Hyperactivity; Hypermethylation; In Vitro; Individual; Isoenzymes; Kinetics; Knowledge; Label; Lead; Link; Literature; Mediating; Methylation; Methyltransferase; Molecular; Mutation; N-terminal; Neurodegenerative Disorders; Nucleic Acid Regulatory Sequences; Oligonucleotides; PHD Finger; Peer Review; Play; Proteins; Publications; Reporting; Research; Research Personnel; Ring Finger Domain; Role; Scanning; Site; Site-Directed Mutagenesis; Specificity; Structure; Students; Tail; Temperature; Tertiary Protein Structure; Titrations; Ubiquitin; Work; biophysical techniques; design; endonuclease; experience; experimental study; gene repression; genetic regulatory protein; genome wide methylation; improved; in vivo; insight; intermolecular interaction; meetings; melting; methylation pattern; mutant; overexpression; prevent; protein expression; protein protein interaction; protein purification; recruit; symposium; training opportunity; undergraduate student

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.

项目摘要 在人类中，最常见的表观遗传学DNA的修饰是细胞学的甲基化，主要是在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（DNMT1）主要负责维持DNA甲基化模式 通过多轮细胞分裂。 DNMT1是具有C末端催化的多域蛋白 甲基转移酶结构域和大型N末端调节区域。复制重点定位 在N末端调节区域发现的序列（RFTS）结构域是DNMT1活性的关键调节剂 体内。 RFTS结构域与DNA结合位点结合，并防止与DNA结合。另外， RFTS结构域参与了几种蛋白质 - 蛋白质相互作用，这些相互作用用于定位和激活DNMT1 催化。最近，已经确定了RFTS结构域中的突变，这导致了两种不同的成年发作 神经退行性疾病。受影响的个体具有全球的修饰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，对这些的生化后果知之甚少 突变。在此提案中，我们试图了解氨基酸序列中的这些变化如何影响 DNMT1的结构和功能。对于特定的目标1，熔融温度和荧光各向异性将 用于检查与疾病相关突变引起的蛋白质稳定性和动力学的变化。 对于特定目标2，将在突变酶中评估RFTS介导的自身抑制作用。两种DNA结合 将在野生型和突变酶中检查亲和力和DNA甲基化活性，以确定是否是否 突变可缓解正常自身抑制作用。对于特定目标3，突变对密钥间分子的影响 将检查互动。已知RFTS结构域与UHRF1结合（类似泛素，包含PHD 和环形域蛋白质1）和修饰的组蛋白H3尾巴。将使用等温滴定量热法 研究RFTS突变对这些调节性蛋白质蛋白质相互作用的影响。我们的初步 工作表明，与疾病相关的突变G589A和V590F降低了蛋白质的热稳定性 还增加了DNA结合亲和力和催化活性，表明至少部分缓解正常的RFTS- 这些突变酶中介导的自身抑制作用。总的来说，这些研究代表了出色的培训 本科生的机会。本科生将参与这项研究的各个方面并获得 实践经验设计实验，收集和分析数据以及解释结果。我们期望 我们的生化研究是为了对疾病相关突变的后果产生关键见解， 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。