NUCLEAR MATRIX STRUCTURE AND DNA REPLICATION

核基质结构和 DNA 复制

• 批准号: 3271959
• 负责人: Ronald Berezney
• 金额: $ 18.16万
• 依托单位: STATE UNIVERSITY OF NEW YORK AT BUFFALO
• 依托单位国家: 美国
• 财政年份: 1977
• 资助国家: 美国
• 起止时间: 1977-09-30 至 1993-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3271959
• 关键词: DNA directed DNA polymerase; DNA primase; DNA replication; DNA topoisomerases; HeLa cells; RNA directed DNA polymerase; affinity chromatography; autoradiography; avidin; biotin; bromodeoxyuridine; cell nucleus; chemical binding; chromatin; chromatography; density gradient ultracentrifugation; electron microscopy; enzyme complex; eukaryote; fluorescence microscopy; gel electrophoresis; immunochemistry; immunofluorescence technique; laboratory rat; liver cells; liver regeneration; nucleic acid inhibitor; nucleic acid metabolism; nucleoproteins; protein sequence; radiotracer; scintillation counter; spectrometry; synchronous cell division; tissue /cell culture; tritium

The overall goal of this project is to elucidate the organizational, functional and regulatory properties of eucaryotic DNA replication in relation to nuclear structure. This area of research is of paramount importance to furthering the understanding of growth and proliferation in normal cells and how these properties are altered in disease states. The proposed research will focus on three basic questions. (1) What are the cell cycle relationships of different replicational components associated with the nuclear matrix? Various properties of DNA polymerase alpha, DNA primase, diadenosine tetraphosphate (AP4A) binding sites, DNA topoisomerase II and other replicative components will be studied at different stages in the cell cycle of synchronized HeLa S3 cells. Particular emphasis will be placed on the possible pre-replicative assembly of these replicative components into multicomponent complexes. (2) What is the structural topography of nuclear matrix-bound replicational components and how does this be performed in mammalian cells grown in monolayers versus nuclear matrix monolayer preparations using antibodies to DNA polymerase alpha, DNA topoisomerase II, 5-bromodeoxyuridine (following in vivo incorporation of BUdR) and fluorescent conjugated avidin following in vitro incorporation of biotin-dUTP. These studies will be performed at different stages in the cell cycle as well as in G1- arrested cells and will then be extended by immunogold labeling to electron microscopic thin sectioning, reinless thick sectioning and whole mount three-dimensional analysis. (3) What are the properties of matrix-bound replicational complexes? Various organizational and functional properties of multi-component replicational complexes solubilized from the nuclear matrix will be studied. For example, we will (1) further purify the complexes by immunoaffinity separation; (2) study the purified complexes by electron microscopy including immunogold localization; (3) determine the polypeptide composition as well as identify specific polypeptides (e.g., DNA polymerase alpha, primase and topoisomerase II) in the released complexes and use this reconstitution system to identify components involved in ATP stimulation of processive DNA synthesis by matrix-bound polymerase alpha.

该项目的总体目标是阐明 真核生物的组织、功能和调节特性 DNA复制与核结构的关系。 该区域的 研究对于加深理解至关重要 正常细胞的生长和增殖以及这些细胞如何生长和增殖 属性在疾病状态下发生改变。 拟议的研究 将重点讨论三个基本问题。 (1)不同细胞周期的关系是什么 与核基质相关的复制成分？ DNA聚合酶α、DNA引物酶的各种特性， 四磷酸二腺苷 (AP4A) 结合位点，DNA 拓扑异构酶 II 和其他复制组件将在不同的地方进行研究 同步 HeLa S3 细胞的细胞周期阶段。 特别的 重点将放在可能的预复制组装上 将这些复制成分分解成多成分复合体。 (2) 核基质结合的结构形貌是怎样的 复制组件以及如何执行 单层生长的哺乳动物细胞与单层核基质细胞 使用 DNA 聚合酶 α、DNA 抗体的制剂 拓扑异构酶 II，5-溴脱氧尿苷（体内遵循 掺入 BUdR) 和荧光共轭亲和素如下 生物素-dUTP 的体外掺入。 这些研究将 在细胞周期的不同阶段以及 G1- 中进行 捕获细胞，然后通过免疫金标记延伸至 电子显微镜薄切片、无束厚切片 以及整体安装三维分析。 (3) 基质束缚复制有什么特点 复合体？ 各种组织和功能属性 溶解的多组分复制复合物 将研究核基质。 例如，我们将 (1) 进一步 通过免疫亲和分离纯化复合物； (2) 研究 通过电子显微镜纯化复合物，包括免疫金 本土化; (3) 确定多肽组成以及 识别特定的多肽（例如 DNA 聚合酶 α、引物酶 和拓扑异构酶 II）在释放的复合物中并使用它 重建系统以识别参与 ATP 的成分 基质结合聚合酶刺激 DNA 持续合成 阿尔法。