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: 10403267
• 负责人: Matt Joseph Jaremko
• 金额: $ 0.25万
• 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/10403267
• 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 基因组复制是所有生命形式的重要事件。 DNA 复制在特定位点启动（称为 复制起点）沿着染色体，以促进基因组的适当复制。在人类中， 调节 DNA 复制起点位置的决定因素相对尚未解决。在这里我们将 从结构上研究建立人类复制起点的关键决定因素，以定义 DNA起源选择机制。建立复制起点的初始因素是起点 识别复合体（ORC）。 ORC 将蛋白质 CDC6 招募到 DNA 复制起点，形成预- 复制复合体（RC 前）。考虑到 ORC 基因的突变，该复合物对于复制至关重要 可能导致有害影响，例如迈尔戈林综合症和不完全导致的癌症 复制。我们将通过结合研究（例如电泳）来研究 ORC•CDC6•DNA 相互作用 迁移率变化 (EMSA) 和福斯特共振能量转移 (FRET) 测定。 ORC•CDC6•DNA 复合体 将通过冷冻电子显微镜（cryo-EM）技术进行进一步分析。研究表明 ORC 和 CDC6 被招募到沿着可变结构染色质的既定位置。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 间歇性定位， 这些核小体影响 ORC 的建立，从而影响复制起点的选择。组蛋白 亚基经历许多翻译后修饰，影响 ORC 与复合物的结合。我们将 通过先进的冷冻电镜方法对预 RC·核小体相互作用进行结构研究，以确定第一个 基因组复制的步骤。预 RC 和核小体重构将被优化以产生稳定的 和同质样品。将开发现代荧光标记方法来分析结合 配合物的性质。翻译后修饰，例如磷酸化和甲基化，将被 旨在确定对 ORC 和核小体识别的影响。结构和结果 结合研究将支持 ChIP-Seq 测定的开发，以绘制 ORC 和复制起点 基因组位置。深入了解 ORC·CDC6·核小体相互作用是解开谜团的关键 DNA 起源选择机制，并将为药物化合物的设计提供见解 逆转不完全复制的影响。我的长期目标是成为一个项目的首席研究员 独立研究实验室，对 DNA 复制的结构生物学进行高影响力的研究 和染色质调节。 NIH F32 奖学金将为以下方面提供巨大的学习和研究支持 这个目标。此外，冷泉港实验室还举办国家会议（例如：真核 DNA 复制和基因组维护）、课程（例如：冷冻电子显微镜，三月）和研讨会 （领导力）对我的科学发展来说是特殊的。