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开放式生物学挑战之一。基因组的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控着生命的基本过程，如可移动的遗传元件(如质粒或转座子)的运动和转录的启动。移动DNA在细菌病原体之间转移，可以传播细菌的致病性(通过毒力基因)，以及耐药性。虽然获得移动DNA只是特定病原体进化的许多阶段中的第一个阶段，如鼠疫、大肠杆菌、霍乱和炭疽病，但已经假设在许多病原体中，共同的机制负责调节细胞间的基因转移。在真核转录中，蛋白质合成的第一步，RNA合成只有在DNA可访问的情况下才能进行：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紧凑状态来控制DNA访问，从而根据需要协调(招募或抑制)转录。移动式DNA转移和染色质组织的许多细节尚不清楚。这项拟议工作的目的是阐明在基因组内移动DNA的转移和组蛋白修饰后的染色质组织中与这种调控相关的结构/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启动相结合，以描述相关的热力学机制。这两个过程都结合了蛋白质结合的超螺旋DNA大系统中受调控的局部蛋白质/DNA相互作用和全球反应。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，在可能的情况下结合宏观特征)，在纳秒尺度上整合原子分辨率的蛋白质/DNA构象，在毫秒尺度上整合超螺旋DNA中位置并置的全球方面。我们的目标是：1)测试/描述假设抑制转座酶单体、二聚体和抑制剂复合体中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DNA大小对超长DNA中位置突触时间和并置机制的影响，从而估计DNA转移的可能性，以补充传统的生化和遗传方法来繁殖微生物毒力；以及3)描述假设的组蛋白H2的两个序列变体和组蛋白H3和H4的修饰尾巴对Chchnatin组织的关键静电效应。由此产生的见解最终可以被用来设计可能限制DNA移动传播从而限制微生物病原体传播的条件，或者影响转录启动，例如调节DNA超级卷曲和核小体组成的酶，以及干扰突触复合体和染色质折叠/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.