NUCLEAR MATRIX STRUCTURE AND DNA REPLICATION

核基质结构和 DNA 复制

• 批准号: 3271956
• 负责人: Ronald Berezney
• 金额: $ 12.77万
• 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 1988-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3271956
• 关键词: DNA directed DNA polymerase; autoradiography; cell nucleus; chromatin; chromatography; density gradient ultracentrifugation; electron microscopy; enzyme complex; eukaryote; gel electrophoresis; immunochemistry; liver cells; liver regeneration; nucleic acid inhibitor; nucleic acid metabolism; radiotracer; scintillation counter; spectrometry; synchronous cell division; tissue /cell culture

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 four basic questions. 1. Is the nuclear matrix involved in the functioning of the matrix-bound DNA polymerase alpha? This question will be addressed with experiments aimed at determining whether the nuclear matrix-bound DNA polymerase alpha enzyme has a greater degree of processivity compared to soluble enzyme preparations. Further experiments will attempt to determine whether increased processivity is a property of the matrix-bound state of the enzyme complex or a special property of the alpha polymerase bound to the matrix. 2. Is the matrix-bound DNA polymerase alpha organized into a replicational complex on the nuclear matrix? As a first step in addressing this question we are developing procedures to release DNA polymerase alpha activity from the nuclear matrix structure. We will then determine whether in vivo replicating DNA is bound to solubilized complexes containing the polymerase activity. 3. What are the properties of the matrix-released complexes? Organizational and functional properties of the released complexes containing DNA polymerase alpha will be studied. For example, we will determine: (1) whether the alpha polymerase in the released complexes continues the synthesis of DNA in vitro along in vivo replicating DNA fragments; (2) whether the alpha polymerase in the released complex has a high degree of processivity; (3) the polypeptide composition as well as identify specific polypeptides of DNA polymerase in the released complex; (4) whether other replicative factors are associated with the released complex; and (5) whether the complexes are dynamically assembled on the matrix pre-replicatively or during in vivo replication. 4. What is the relationship of these matrix-bound replicational complexes to chromatin structure? As a first step in addressing this question we will determine whether DNA is attached to the nuclear matrix in a nucleosomal repeat structure. We will then attempt to isolate replicational complexes containing matrix-attached chromatin fragments and study the structural organization and functional properties of these putative replicational complexes.

该项目的总体目标是阐明组织、 真核生物 DNA 复制的功能和调控特性 与核结构的关系。 这一领域的研究至关重要 加深对增长和扩散的理解的重要性 正常细胞以及这些特性在疾病状态下如何改变。 这 拟议的研究将集中于四个基本问题。 1. 核基质是否参与基质结合的功能？ DNA聚合酶α？ 这个问题将通过实验来解答 旨在确定核基质结合的DNA聚合酶α是否 与可溶性酶相比，酶具有更大程度的持续加工能力 准备工作。 进一步的实验将尝试确定是否 增加的持续合成能力是矩阵束缚态的一个属性 酶复合物或与α聚合酶结合的特殊性质 矩阵。 2. 基质结合的 DNA 聚合酶 α 是否组织成复制体？ 核基质上的复合物？ 作为解决这个问题的第一步 我们正在开发释放 DNA 聚合酶 α 活性的程序 核基质结构。 然后我们将确定是否在体内 复制 DNA 与含有聚合酶的溶解复合物结合 活动。 3. 基质释放复合物有哪些特性？ 释放复合物的组织和功能特性 将研究含有DNA聚合酶α的物质。 例如，我们将 确定：（1）释放的复合物中是否存在α聚合酶 继续体外 DNA 合成以及体内 DNA 复制 碎片； (2)释放的复合物中的α聚合酶是否具有 高度的持续性； (3)多肽组合物以及 鉴定释放的复合物中DNA聚合酶的特定多肽； (4)是否与释放的其他复制因素有关 复杂的; (5)复合物是否动态组装在 复制前或体内复制期间的基质。 4. 这些基质结合复制复合物之间的关系是什么 染色质结构？ 作为解决这个问题的第一步，我们 将确定 DNA 是否附着在核基质上 核小体重复结构。 然后我们将尝试隔离 含有基质附着的染色质片段的复制复合物和 研究这些的结构组织和功能特性 假定的复制复合体。