DNA Supercoiling: Local and Global Aspects

描述（由申请人提供）：了解染色体组织及其对基因表达的控制是最基本的生物学挑战之一。基因组的组织和表达是密切相关的，因为DNA和蛋白质结合DNA的复杂结构和动态在大范围的空间和时间尺度上调节着生命的基本过程，如质粒或转座子等移动遗传元件（“移动DNA”）的运动和转录起始。移动dna在细菌病原体之间转移，可以传播细菌致病性（通过毒力基因）以及耐药性。虽然获得可移动dna只是特定病原体（如鼠疫、大肠杆菌、霍乱和炭疽）进化的许多阶段中的第一个阶段，但人们已经假设，在许多病原体中，有共同的机制负责调节细胞间基因转移。在真核生物转录（蛋白质合成的第一步）中，RNA合成只能在DNA可接近时进行：通过核小体修饰、变异组蛋白和重塑的复杂网络，假设真核生物基因组改变染色质纤维的折叠和压实状态，以控制DNA的进入，并根据需要协调（招募或抑制）转录。许多移动DNA转移和染色质组织的细节是未知的。提出的工作的目标是阐明在组蛋白修饰后基因组内移动dna转移和染色质组织中与这种调控控制相关的结构/动力学机制。我们的长期目标是将染色质组织和调控的结构和动力学方面与转录起始结合起来，以描述所涉及的热力学机制。这两个过程结合了调节局部蛋白质/DNA相互作用和蛋白质结合超卷曲DNA大系统的全局反应。基于先前支持下完成的模型和应用，我们将通过中尺度模型（在需要的地方结合局部细节，在可能的地方结合宏观特征），将纳秒尺度上原子分辨率的蛋白质/DNA构象与毫秒尺度上超螺旋DNA中位点并立的全局方面结合起来。我们的目标是：1)测试/描述假设抑制特定转座酶单体，二聚体和抑制剂复合物中的转座的局部构象变化；2)确定质粒超螺旋度、DNA缠绕构象和移动DNA大小对位点突触时间和超长DNA并置机制的影响，从而估计DNA转移的概率，以补充传统的生化和遗传方法来传播微生物毒力；3)描述组蛋白H2的两个序列变体和组蛋白H3和H4的修饰尾部对染色质组织的关键静电效应的假设。由此产生的见解最终可以用于设计可能限制移动DNA繁殖和微生物病原体传播的条件，或影响转录起始的条件，例如调节DNA超卷曲和核小体组成的酶，以及干扰突触复合物和染色质折叠/展开重排的药物化合物。

DESCRIPTION (provided by applicant): Understanding chromosome organization and its control of gene expression represents one of the most fundamental open biological challenges. Genomic organization and expression are intimately related because the complex structure and dynamics of DNA and protein-bound DNA at a large range of spatial and temporal scales regulate basic processes of life, such as the movement of mobile genetic elements ("mobile DNA") like plasmids or transposons, and transcription initiation. Mobile DNAs are transferred among bacterial pathogens and can propagate bacterial pathogenicity (through virulence genes), as well as drug resistance. While the acquisition of mobile DNAs is only the first of many stages in the evolution of specialized pathogens such as plague, E. coli, cholera, and anthrax, it has been hypothesized that common mechanisms are responsible for regulating intercellular gene transfer in many pathogens. In eukaryotic transcription, the first step in protein synthesis, RNA synthesis can only proceed when the DNA is accessible: through a complex network of nucleosome modifications, variant histones, and remodeling, it is hypothesized that eukaryotic genomes alter states of folding and compaction of the chromatin fiber to control DNA access and, as such, orchestrate (recruit or repress) transcription as needed. Many details of mobile DNA transfer and chromatin organization are unknown. The goal of the proposed work is to elucidate structural/dynamical mechanisms associated with such regulatory control in the transfer of mobile DNAs within genomes and in chromatin organization following histone modifications. Our long-term goals are to integrate structural and dynamics aspects of chromatin organization and regulation with transcription initiation to delineate thermodynamic mechanisms involved. Both processes combine regulatory local protein/DNA interactions with global responses in large systems of protein-bound supercoiled DNA. Based on models and applications completed under prior support, we will integrate protein/DNA conformations at atomic resolution on the nanosecond scale with global aspects of site juxtaposition in supercoiled DNA on the millisecond scale through mesoscale models (which incorporate local details where needed and macroscopic features where possible). Our aims are to: 1) test/delineate the local conformational changes hypothesized to repress transposition in specific transposase monomer, dimer, and inhibitor complexes; 2) determine effects of plasmid superhelicity, DNA interwound conformations, and mobile DNA size on site synapsis times and juxtaposition mechanisms in very long DNA, and thereby estimate the probability of DNA transfer to complement traditional biochemical and genetic approaches to propagation of microbial virulence; and 3) delineate hypothesized crucial electrostatic effects of two sequence variants of histone H2 and modified tails of histones H3 and H4 on chrornatin organization. Resulting insights can ultimately be exploited to design conditions that might limit mobile DNA propagation and hence microbial pathogen spread, or affect transcription initiation, such as enzymes that regulate DNA super coiling and nucleosome composition and pharmaceutical compounds that interfere with synaptic complexes and chromatin folding/unfolding rearrangements.