NUCLEAR MATRIX STRUCTURE AND DNA REPLICATION

本项目的总体目标是阐明组织， 真核生物DNA复制的功能和调节特性 与核结构的关系。 这个研究领域至关重要 重要性，以进一步了解增长和扩散， 正常细胞以及这些特性在疾病状态下如何改变。 的 拟议的研究将集中在四个基本问题上。 1.核基质是否参与了基质结合的功能 DNA聚合酶α？ 这个问题将通过实验来解决 旨在确定核基质结合的DNA聚合酶α 与可溶性酶相比，酶具有更大程度的持续合成能力 准备工作 进一步的实验将试图确定是否 增加的持续合成能力是基质结合状态的性质， 酶复合物或结合到酶复合物的α聚合酶的特殊性质。 矩阵 2.结合在基质上的DNA聚合酶α是否被组织成一个复制的 核基质上的复合体 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活性的方法， 核矩阵结构。 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.基质释放复合物的性质是什么？ 释放复合物的组织和功能特性 将研究含有DNA聚合酶α的细胞。 比如我们会 确定：（1）释放的复合物中的α聚合酶是否 继续沿着体内复制DNA的体外DNA合成 （2）释放的复合物中的α聚合酶是否具有与α聚合酶的结合位点。 高度的持续合成能力;（3）多肽组合物以及 鉴定释放的复合物中DNA聚合酶的特异性多肽; (4)其他复制因子是否与释放的 复合物;以及（5）复合物是否在 基质复制前或体内复制期间。 4.这些结合在基质上的复制复合体之间的关系是什么 与染色质结构的关系 作为解决这个问题的第一步， 将决定DNA是否附着在核基质上， 核小体重复结构 然后我们将尝试分离 含有基质附着的染色质片段的复制复合物， 研究这些的结构组织和功能特性， 假定的复制复合体

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.