DNA TOPOISOMERASE II AND CHROMOSOME STRUCTURE

DNA 拓扑异构酶 II 和染色体结构

• 批准号: 6239945
• 负责人: Scott Matthew Williams
• 金额: $ 9.68万
• 依托单位: MEHARRY MEDICAL COLLEGE
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-08-01 至 1998-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6239945
• 关键词: DNA binding protein; DNA replication; DNA topoisomerases; Drosophilidae; active sites; chromatin; chromosomes; clone cells; computer assisted sequence analysis; enzyme inhibitors; eukaryote; heterochromatin; molecular cloning; nucleic acid sequence; nucleic acid structure; protein structure function; pulsed field gel electrophoresis; radionuclides; southern blotting

The long-term goal of this research is to determine the underlying biochemical/genetic properties that differentiate the DNA on eukaryotic chromosomes into its distinct classes - euchromatin and heterochromatin. Euchromatin and heterochromatin differ in very fundamental characteristics with euchromatin containing genes that have distinct phenotypic effects, and the heterochromatin, originally defined cytologically as highly condensed DNA, being a repository of repeated, non-coding DNA sequences. Although heterochromatic DNA has largely unknown function, it is essential for the normal division of the genetic material during meiosis and mitosis. It is therefore likely that some or most heterochromatic DNA is not "junk" but rather a collection of sequences that function via non-transcriptional mechanisms. This research will elucidate one aspect of this repeated DNA that has been proposed as important in causing heterochromatin to behave as it does - interactions with topoisomerase II (topo II). One hypothesis proposes that heterochromatic behavior is a function of its interactions with the DNA modifying protein, topo II. Topo II, an important component of the nuclear matrix in Drosophila, has been shown to be important in many of the features usually associated with heterochromatin from DNA condensation to affecting the frequency of recombination. Topo II poisons are also potent antitumor drugs. Preliminary evidence indicates that heterochromatin is highly enriched for sites essential for topo II function, leading to the hypothesis that chromatin structure can be explained by the differing density of topo II sites. This hypothesis is primarily based on conceptualization of DNA sequences extracted from the GenBank database. Therefore, to fully test this idea it is necessary to determine the generality of the observation. This will be done using two experimental approaches. First, clones of the sequences subjected to computer analysis will be used for in vitro analysis to test for topo II binding and cutting. Clones from heterochromatic regions should have a higher density of legitimate topo II sites. Second, the distribution of functional topo II sites in euchromatin and heterochromatin will be compared in vivo using cell lines. A topo II inhibitor, VP-16, that causes easily discernible topo II cleavage will be applied to Drosophila cell cultures and DNA will then be fractionated as a function of topo II cleavage. Southern blots of the DNA from inhibitor treated cells from both standard and pulse field gels will be probed with large PI clones from euchromatic and heterochromatic locations. The hypothesis predicts that heterochromatic regions will contain many more active topo II sites as determined by the number of cleavable sites per kilobase.

这项研究的长期目标是确定基础 在真核上区分DNA的生化/遗传特性 染色体变成其独特的类 - 白染色质和 异染色质。 白染色质和异染色质在非常不同 含有共染色质的基本基因的基本特征 最初 在细胞学上定义为高度凝结的DNA，是 重复的非编码DNA序列。 虽然异型DNA具有 在很大程度上未知的功能，对于正常分裂至关重要 减数分裂和有丝分裂期间的遗传物质。 因此是 某些或大多数异质的DNA可能不是“垃圾”，而是 通过非转录功能运行的序列集合 机制。 这项研究将阐明这一重复的一个方面 提出在引起异染色质的DNA至关重要的DNA 与拓扑异构酶II相互作用（TOPO II）。 一个假设提出异质性行为是 它与DNA修饰蛋白的相互作用Topo II。 Topo II，AN 已经显示了果蝇中核基质的重要组成部分 在许多通常与之相关的功能中很重要 从DNA凝结到影响频率的异染色质 重组。 Topo II毒药也是有效的抗肿瘤药物。 初步证据表明异染色质高度富集 对于Topo II功能必不可少的站点，导致假设 染色质结构可以通过不同的密度来解释 TOPO II站点。 该假设主要基于概念化 从GenBank数据库中提取的DNA序列的。 因此，要 充分测试这个想法，有必要确定 观察。这将使用两种实验方法完成。 首先，经过计算机分析的序列的克隆将是 用于体外分析，用于测试TOPO II结合和切割。 异质区域的克隆应具有更高的密度 合法的Topo II站点。 其次，功能性拓扑的分布 将在体内比较白染色质和异染色质中的II位点 使用单元线。 TOPO II抑制剂VP-16，很容易引起 可识别的Topo II裂解将应用于果蝇细胞 然后，培养物和DNA将作为Topo II的函数分馏 乳沟。 来自抑制剂处理过的细胞的DNA的南部印迹 标准凝胶和脉冲场凝胶都将用大型PI克隆进行探测 从圣体和异，位置。 假设 预测异质区域将包含更多活跃的区域 由可裂解位点的数量确定的TOPO II站点 千个酶。