Supercoiling in genome topology and transcription

项目总结 DNA超螺旋是基因组中普遍存在的一种特征，由转运酶和 通过许多蛋白质的结合，这些蛋白质包裹或改变它们所结合的序列的扭曲。因此，基因组学 超螺旋是动态的，是基于基因组的活动的敏感调节器，如转录和 重组。最近的大多数努力都集中在转录生成的超级卷曲，即孪生结构域 模型，以及拓扑异构酶的活性。相反，在很大程度上没有被探索的是DNA变化的影响 在细胞生理学的主要方面，例如(I)远距离基因组相互作用和(Ii) 建筑蛋白与DNA的结合。绘制基因组超螺旋图谱的一个令人满意的策略(III)也是 缺乏，因为目前的方法利用的是改变双螺旋结构的探针。因此， 利用我们在磁镊子、蛋白质介导的DNA环、类核相关蛋白质等方面的专业知识， 在体外和体内转录检测方面建立了良好的合作关系，我们已经制定了目标 将极大地提高我们在以下三个基本领域的知识： (1)检验DNA超螺旋显著促进拓扑结构形成的假设， 例如蛋白质介导的环路。使用lac阻遏蛋白(Laci)作为DNA环蛋白，一系列 环长，以及在体外和体内的互补试验，我们将建立超螺旋水平， 张力和类核相关蛋白，最有可能在体内催化环路。 (2)检验DNA超螺旋影响蛋白质结合的假设，这些蛋白质定义了 基因组。我们将建立(I)解离常数和(Ii)DNA的依赖于DNA拓扑 由代表性的、丰富的核相关蛋白(NAP)非特异性地结合DNA而致密化。 (3)检验启动子活性受距离和超螺旋影响的假设 方式，通过上游蛋白质介导的环。

PROJECT SUMMARY DNA supercoiling is a ubiquitous feature of genomes, generated by the activity of translocating enzymes and by the binding of many proteins that wrap or change the twist of sequences to which they bind. Thus, genomic supercoiling is dynamic and is a sensitive regulator of genome-based activities, such as transcription and recombination. Most recent efforts have focused on transcription-generated supercoiling, the twin domain model, and the activity of topoisomerases. Largely unexplored is instead the impact of changes in DNA supercoiling on major aspects of cell physiology such as (i) long distance genomic interactions and (ii) the binding of architectural proteins to DNA. A satisfactory strategy to map genomic supercoiling (iii) is also lacking, since current approaches utilize probes that alter the structure of the double helix. Therefore, leveraging our expertise with magnetic tweezers, protein-mediated DNA looping, nucleoid associated proteins, and well established collaborations for in vitro and in vivo transcription assays, we have developed aims that will advance significantly our knowledge in these three fundamental areas: (1) Test the hypothesis that DNA supercoiling significantly facilitates the formation of topological structures, such as protein-mediated loops. Using the lac repressor protein (LacI) as a DNA looping protein, a range of loop lengths, and complementary in vitro and in vivo assays, we will establish the levels of supercoiling, tension and nucleoid associated proteins which most likely catalyze in vivo looping. (2) Test the hypothesis that DNA supercoiling affects the binding of proteins that define the architecture of the genome. We will establish the dependence on DNA topology of the (i) dissociation constant, and (ii) DNA compaction by representative, abundant nucleoid associated proteins (NAPs) that bind DNA non-specifically. (3) Test the hypothesis that promoter activity is affected, in a distance- and supercoiling-dependent manner, by an upstream protein-mediated loop.