Structural investigation of human ORC: a key determinant of DNA origin selection

人类 ORC 的结构研究：DNA 起源选择的关键决定因素

• 批准号: 10057386
• 负责人: Matt Joseph Jaremko
• 金额: $ 6.86万
• 依托单位: COLD SPRING HARBOR LABORATORY
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-12-01 至 2021-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10057386
• 关键词: Address; Affinity; Award; Basal Cell Nevus Syndrome; Binding; Binding Sites; Biological Assay; Biological Sciences; CDC6 gene; Cell Cycle; ChIP-seq; Chromatin; Chromatin Structure; Chromosomes; Cloning; Complex; Cryoelectron Microscopy; DNA; DNA Binding; DNA biosynthesis; DNA replication origin; Development; Disease; Dwarfism; Educational workshop; Electrophoretic Mobility Shift Assay; Environment; Event; Fellowship; Fluorescence Resonance Energy Transfer; Generations; Genes; Genetic Transcription; Genome; Genomic Instability; Goals; Histones; Human; Investigation; Label; Laboratories; Laboratory Research; Lead; Leadership; Learning; Length; Licensing; Life; Link; Location; Maintenance; Malignant Neoplasms; Maps; Methods; Methylation; Modernization; Modification; Molecular; Molecular Analysis; Mutation; Nucleosomes; Oncogenic; Pharmacologic Substance; Phosphorylation; Positioning Attribute; Post-Translational Protein Processing; Pre-Replication Complex; Principal Investigator; Property; Proteins; Regulation; Replication Initiation; Replication Origin; Research; Research Support; Research Training; Resolution; Roentgen Rays; Sampling; Site; Structure; Techniques; Technology; Titan; Training; United States National Institutes of Health; Work; Writing; base; career development; design; improved; insight; laboratory curriculum; meetings; origin recognition complex; programs; protein expression; reconstitution; recruit; scaffold; structural biology; symposium; tool; training opportunity

Abstract Genome replication is an essential event in all forms of life. DNA replication is initiated at specific sites (termed origins of replication) along chromosomes to facilitate appropriate duplication of the genome. In humans, the determinants that regulate the location of DNA replication origin are relatively unresolved. Here we will structurally investigate key determinants that establish origins of replication in humans to define the mechanism of DNA origin selection. The initial factor that establishes the origin of replication is the origin recognition complex (ORC). ORC recruits the protein CDC6 to the DNA origin of replication to form the pre- replicative complex (pre-RC). The complex is essential for replication, considering mutations in ORC genes can lead to deleterious effects, such as Meier-Gorlin Syndrome and cancer resulting from incomplete replication. We will investigate ORC•CDC6•DNA interactions through binding studies, such as electrophoretic mobility shift (EMSA) and Förster resonance energy transfer (FRET) assays. The ORC•CDC6•DNA complex will be further analyzed through cryoelectron microscopy (cryo-EM) techniques. Studies have shown ORC and CDC6 are recruited to established locations along variably structured chromatin. The chromatin regions of active transcription consist of histone complexes, called nucleosomes, positioned intermittently along DNA and these nucleosomes influence ORC establishment and therefore replication origin selection. The histone subunits undergo many posttranslational modifications that influence ORC binding to the complex. We will structurally investigate pre-RC•nucleosome interactions through advanced cryo-EM methods to define the first step in genome replication. The pre-RC and nucleosome reconstitution will be optimized to generate stable and homogenous samples. Modern fluorescent labelling-approaches will be developed to analyze the binding properties of the complexes. Posttranslational modifications, such as phosphorylation and methylation, will be addressed to determine the influence on ORC and nucleosome recognition. The results from the structural and binding studies will support development of a ChIP-Seq assay to map out the ORC and replication origin genome location. An in depth understanding of ORC•CDC6•nucleosome interactions are key to unraveling the mechanism of DNA origin selection and will provide insight for the design of pharmaceutical compounds that reverse the effects of incomplete replication. My long-term goal is to become the principal investigator of an independent research laboratory that conducts high impact studies on the structural biology of DNA replication and chromatin regulation. The NIH F32 fellowship will provide immense learning and research support towards this goal. In addition, the Cold Spring Harbor Laboratory harbors national meetings (ex: Eukaryotic DNA Replication & Genome Maintenance), courses (ex: Cryoelectron Microscopy, March), and workshops (Leadership) that are exceptional for my scientific development.

抽象的 基因组复制是各种生活形式的重要事件。 DNA复制是在特定位点启动的（称为 复制的起源）沿染色体促进基因组的适当重复。在人类中 调节DNA复制来源位置的决定因素相对未解决。我们会在这里 结构研究密钥确定建立人类复制的起源以定义 DNA起源选择机理。建立复制起源的初始因素是原点 识别综合体（ORC）。 ORC将蛋白Cdc6招募到复制的DNA起源，以形成前 复制复合物（PRE-RC）。该复合物对于复制至关重要，考虑了兽人基因中的突变 可能导致有害影响，例如Meier-Gorlin综合征和不完整的癌症 复制。我们将通过结合研究（例如电泳）研究ORC•CDC6•DNA相互作用 移动性转移（EMSA）和Förster共振能量转移（FRET）测定法。 ORC•CDC6•DNA复合物 将通过低温电子显微镜（Cryo-EM）技术进一步分析。研究表明兽人和 CDC6沿着可变结构化的染色质募集到已建立的位置。染色质区域 活性转录由组蛋白复合物（称为核小体）组成，沿着DNA和 这些核体影响ORC的建立，因此会影响复制起源选择。组蛋白 亚基经历了影响ORC与复合物结合的许多翻译后修饰。我们将 结构研究前RC•通过晚期冷冻EM方法的核小体相互作用来定义第一个 介入基因组复制。前RC和核体重建将优化以产生稳定 和同质样品。将开发现代荧光标签 - 接收器来分析结合 复合物的特性。翻译后修饰（例如磷酸化和甲基化）将是 确定对ORC和核小体识别的影响。结构和 绑定研究将支持开发芯片seq分析，以绘制兽人和复制起源 基因组位置。对ORC•CDC6的深入了解•核小体相互作用是揭开核心体的关键 DNA起源选择的机理，并将为设计化合物的设计提供见解 扭转不完整复制的影响。我的长期目标是成为 对DNA复制的结构生物学进行高影响研究的独立研究实验室 和染色质调节。 NIH F32奖学金将为 这个目标。此外，冷春港实验室拥有全国会议（例如：真核DNA 复制和基因组维护），课程（例如：冷冻电子显微镜，3月）和讲习班 （领导力）对我的科学发展非常出色。