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代表了最基本的开放生物学挑战之一。 基因组组织和表达密切相关，因为DNA和蛋白质结合的DNA在大范围的空间和时间尺度上的复杂结构和动力学调节生命的基本过程，例如移动的遗传元件（“移动的DNA”）如质粒或转座子的移动，以及转录起始。 移动的DNA在细菌病原体之间转移，并且可以传播细菌致病性（通过毒力基因）以及耐药性。 虽然获得移动的DNA只是鼠疫等特殊病原体进化过程中许多阶段的第一步，但E.大肠杆菌、霍乱和炭疽，已经假设共同的机制负责调节许多病原体中的细胞间基因转移。 在真核转录中，蛋白质合成的第一步，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，假设真核基因组改变染色质纤维的折叠和压缩状态以控制DNA接近，因此，根据需要协调（招募或抑制）转录。 移动的DNA转移和染色质组织的许多细节是未知的。 拟议的工作的目标是阐明结构/动力学机制与这样的监管控制在基因组内的移动的DNA的转移和组蛋白修饰后的染色质组织。 我们的长期目标是整合结构和动力学方面的染色质组织和转录起始的调控，描绘热力学机制参与。 这两个过程结合了联合收割机调节局部蛋白质/DNA相互作用与蛋白质结合的超螺旋DNA的大系统中的全局响应。 基于先前支持下完成的模型和应用程序，我们将整合蛋白质/DNA构象在原子分辨率的纳秒级与全球方面的网站并列超螺旋DNA在毫秒级通过中尺度模型（其中包括本地的细节需要和宏观功能在可能的情况下）。 我们的目标是：1）测试/描绘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）确定质粒超螺旋性、DNA缠绕构象和移动的DNA大小对极长DNA中的位点突触时间和并置机制的影响，从而估计DNA转移的可能性，以补充传统的生物化学和遗传方法来繁殖微生物毒力;和3）描绘了组蛋白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假设的关键静电效应。由此产生的见解最终可以被利用来设计可能限制移动的DNA繁殖并因此限制微生物病原体传播或影响转录起始的条件，例如调节DNA超螺旋和核小体组成的酶以及干扰突触复合物和染色质折叠/解折叠重排的药物化合物。

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.