NUCLEAR MATRIX STRUCTURE AND DNA REPLICATION

这个项目的总体目标是阐明 真核生物的组织、功能和调控特性 DNA复制与核结构的关系。这一地区 研究对于加深理解是至关重要的 在正常细胞中的生长和增殖以及这些 在疾病状态下，属性会发生变化。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成分？ DNA聚合酶α，DNA底物酶， 二腺苷四磷酸(AP4A)结合部位，DNA拓扑异构酶 II和其他复制成分将在不同的 同步培养的HeLa S3细胞的细胞周期分期。特例 重点将放在可能的预复制组装上 将这些可复制成分转化为多组分复合体。 (2)核基质结合的结构形貌是什么？ 复制组件以及如何在 生长在单层中的哺乳动物细胞与核基质单层 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(体内观察 BUDR)和荧光偶联亲和素的掺入 生物素-dUTP的体外掺入。这些研究将是 在细胞周期的不同阶段以及在G1- 然后将通过免疫金标记法扩展到 电子显微镜薄切片、无筋厚切片 并进行整体安装的三维分析。 (3)矩阵限制复制的性质是什么 情结？的各种组织和功能特性 从增溶的多组分复制络合物 将研究核基质。例如，我们将(1)进一步 用免疫亲和分离法纯化配合物；(2)研究 包括免疫金在内的电子显微镜下纯化的复合体 定位；(3)测定多肽组成以及 识别特定的多肽(例如，DNA聚合酶α、启动酶 和拓扑异构酶II)，并使用此 识别三磷酸腺苷中涉及的成分的重构系统 基质结合聚合酶对DNA合成的刺激作用 阿尔法。

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.