NUCLEAR MATRIX STRUCTURE AND DNA REPLICATION

核基质结构和 DNA 复制

• 批准号: 3271953
• 负责人: Ronald Berezney
• 金额: $ 16.6万
• 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/3271953
• 关键词: DNA directed DNA polymerase; 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 桉树的组织，功能和法规特性 与核结构有关的DNA复制。 这个区域 研究对于促进理解至关重要 正常细胞的生长和增殖以及如何 疾病状态的特性发生了改变。 拟议的研究 将重点关注三个基本问题。 （1）不同的细胞周期关系是什么 与核基质相关的复制组件？ DNA聚合酶α的各种特性，DNA Primase， 十二生四磷酸（AP4A）结合位点，DNA拓扑异构酶 II和其他重复组件将在不同的 同步HELA S3细胞的细胞周期中的阶段。 特别的 将重点放在可能的复制前组装上 这些复制成分中的多组分复合物。 （2）核基质结合的结构地形是什么 复制组件以及如何在 在单层中生长的哺乳动物细胞与核基质单层 使用抗DNA聚合酶α，DNA的制剂 拓扑异构酶II，5-溴脱氧尿苷（在体内遵循 结合BUDR）和荧光偶联的Avidin之后 体外掺入生物素-DUTP。 这些研究将是 在细胞周期以及G1-中的不同阶段进行 逮捕的细胞，然后通过免疫金标记将其扩展到 电子显微镜薄截面，厚厚的截面 和整个安装三维分析。 （3）矩阵结合复制的属性是什么 复合物？ 各种组织和功能属性 多组分复制络合物从 将研究核矩阵。 例如，我们将（1）进一步 通过免疫亲和力分离净化复合物； （2）研究 通过电子显微镜纯化的复合物，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，底漆 和拓扑异构酶ii）在已释放的复合物中使用并使用此功能 重建系统以识别与ATP有关的组件 通过基质结合聚合酶刺激过程DNA合成 阿尔法。