NUCLEAR MATRIX STRUCTURE AND DNA REPLICATION

本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 的这个区域 研究对于进一步了解 正常细胞的生长和增殖， 在疾病状态下，属性会发生改变。 拟议研究 将集中讨论三个基本问题。 (1)不同的细胞周期之间的关系是什么 与核基质相关的复制组件 DNA聚合酶α，DNA引发酶， 二腺苷四磷酸（AP 4A）结合位点，DNA拓扑异构酶 II和其他复制组件将在不同的研究 同步化的HeLa S3细胞的细胞周期中的阶段。 特别 重点将放在可能的预复制组装上 将这些复制成分转化为多成分复合物。 (2)什么是核基质结合的结构拓扑 复制组件，以及如何在 单层哺乳动物细胞与核基质单层细胞 使用DNA聚合酶α，DNA 拓扑异构酶II，5-溴脱氧尿苷（体内 掺入BUdR）和荧光缀合的抗生物素蛋白 生物素-dUTP的体外掺入。 这些研究报告将 在细胞周期的不同阶段以及在G1- 然后通过免疫金标记将其延伸， 电子显微镜薄切片，无筋厚切片 和整体三维分析。 (3)矩阵约束复制的性质是什么 情结？ 的各种组织和功能特性 多组分复制复合物溶解于 核矩阵将被研究。 例如，我们将（1）进一步 免疫亲和分离纯化复合物;（2）研究复合物的性质。 通过电子显微镜纯化的复合物，包括免疫金 定位;（3）确定多肽组成以及 鉴定特定多肽（例如，DNA聚合酶α 和拓扑异构酶II），并使用这种 重组系统，以识别参与ATP的组分 基质结合聚合酶对DNA合成促进作用 α的

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.