DNA-Protein Interactions at the Replication Fork

复制叉处的 DNA-蛋白质相互作用

• 批准号: 7582715
• 负责人: JACK D GRIFFITH
• 金额: $ 33.89万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 1983
• 资助国家: 美国
• 起止时间: 1983-04-01 至 2012-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7582715
• 关键词: Anti-Bacterial Agents; Architecture; Area Analyses; Bacteria; Binding; Binding Proteins; Biochemical; Biological Assay; Buffers; Cell Extracts; Cells; Chromosomes; Collaborations; Complex; Coupled; Coupling; Cryoelectron Microscopy; DNA; DNA Primase; DNA biosynthesis; DNA-Protein Interaction; Data; Development; Drug Delivery Systems; Electron Microscopy; Elements; Eukaryotic Cell; Funding; Genetic Recombination; Goals; Grant; Herpes Simplex Infections; Herpesvirus 1; Homologous Gene; Human; Human Papillomavirus; Imagery; In Vitro; Incubated; Laboratories; Lead; Magnetism; Mechanics; Methods; Modeling; Nature; Pharmaceutical Preparations; Polymerase; Postdoctoral Fellow; Pre-Replication Complex; Procedures; Protein Biochemistry; Proteins; Rec A Recombinases; Replication Origin; Research Personnel; Resolution; Resources; Simplexvirus; Structure; Superhelical DNA; System; TP53 gene; Techniques; Technology; Time; Training; Viral; Virus; Visit; Work; Yeasts; design; drug development; fungus; helicase; image reconstruction; magnetic beads; member; nanoscale; new technology; origin recognition complex; particle; programs; protein complex; protein structure; public health relevance; reconstruction; single molecule; tool

DESCRIPTION (provided by applicant): Analysis of single molecules by EM provides a powerful approach to study the mechanics of DNA replication. The long-term focus of this study combines biochemistry, protein tagging, with direct visualization of DNA protein complexes by electron microscopy (EM). A statistically large number of molecules are examined and this information is combined with data from biochemical assays. This project has great strength in its long standing interactions with Dr. Steve Bell (yeast ORC), Drs. Tom Broker and Louise Chow (human papillomaviruses (HPV)), and Dr. Charles Richardson (T7 replication). These studies are augmented with work carried out in this laboratory on T4 and Herpes Simplex type 1 (HSV-1). The goal is to understand origin activation (HPV, yeast) and the architecture of a moving fork (T7, T4, HSV-1). A new technology using nano-scale paramagnetic particles attached to specifically designed DNA templates allows visualization of nanogram quantities of DNA and protein and the rapid exchange DNA-protein complexes into buffers optimal for high resolution EM. DNAs containing the yeast ARS1 element attached to magnetic particles will be incubated with yeast cell extracts to load the pre-RC complex including Mcm and Cdc6 factors. Their structure will be probed by Western analysis and EM using nanoscale 'biopointers'. DNA fragments containing the HPV origin bound to paramagnetic particles will be assembled with the E1, E2 proteins as well as factors from 293 cell extracts. The stepwise opening of the origin and its interaction with cell factors including p53 will be investigated using a combination of EM, Western analysis, and replication assays. Continued studies of the moving fork in T7 and T4 will employ paramagnetic particles along with single particle image reconstructions and cryoEM to generate high resolution structures containing the polymerase, helicase-primase, SSBs and other factors on model replication forks. Using HSV-1 replication proteins purified in this laboratory, the architecture of DNA-protein complexes which catalyze replication will be examined to provide information on the looping of the lagging strand in a moving eukaryotic fork. Using these in vitro T4 and HSV-1 replication systems, the way in which recombination coupled replication is initiated by recombination factors and how the replication forks proceed to generate networks of replicating DNA will be visualized. Understanding the structure of the proteins and protein complexes which catalyze replication of prokaryotic and eukaryotic chromosomes and viruses is crucial to the development of drugs which bind key components at replication forks. This will help lead to development of new anti-bacterial, fungal, and viral compounds. PUBLIC HEALTH RELEVANCE Understanding the structure of the proteins and protein complexes which catalyze replication of prokaryotic and eukaryotic chromosomes and viruses is crucial to the rational design of new drugs which bind these proteins and structures. This study employs high resolution electron microscopy to provide a unique and direct visualization of protein complexes at replication origins and replication forks in bacteria, fungi, eukaryotic cells and viruses to provide the information needed to identify new drug targets.

描述（由申请人提供）：EM对单分子的分析提供了一种研究DNA复制力学的强大方法。这项研究的长期重点结合了生物化学，蛋白质标记，以及通过电子显微镜（EM）直接可视化DNA蛋白复合物。检查了大量分子，并将此信息与生化测定的数据相结合。该项目在与史蒂夫·贝尔（Steve Bell）（酵母兽人）博士（Drs）的长期互动中具有很大的力量。汤姆经纪人和路易丝·乔（Human Papillomaviruess（HPV））和查尔斯·理查森（Charles Richardson）博士（T7复制）。这些研究通过在该实验室对T4和单纯疱疹1型（HSV-1）进行的工作进行了增强。目的是了解原点激活（HPV，酵母）和移动叉的架构（T7，T4，HSV-1）。一项使用连接到专门设计的DNA模板的纳米尺度顺磁性颗粒的新技术允许可视化DNA和蛋白质的纳米图量，以及快速交换DNA-蛋白质复合物为最佳的缓冲液，用于高分辨率EM。含有连接到磁性颗粒的酵母ARS1元件的DNA将与酵母细胞提取物一起孵育，以加载PER-RC复合物，包括MCM和CDC6因子。它们的结构将通过西方分析探测，并使用纳米级“生物启动器”来探测它们的结构。包含与顺磁性颗粒结合的HPV起源的DNA片段将与E1，E2蛋白以及来自293个细胞提取物的因子组装在一起。原点的逐步开放及其与包括p53在内的细胞因子的相互作用将使用EM，西方分析和复制测定的组合进行研究。对T7和T4中移动叉的持续研究将采用顺磁性颗粒以及单个粒子图像重建和冷冻剂，以生成含有聚合酶，解旋酶 - 蛋白酶，SSB，SSB和其他因素的高分辨率结构，以及模型复制叉上的其他因素。使用在该实验室纯化的HSV-1复制蛋白，将检查DNA蛋白复合物的结构，这些蛋白的结构将被检查，以提供有关移动的真核叉中滞后链环绕的信息。使用这些体外T4和HSV-1复制系统，将通过重组因子启动重组耦合复制的方式以及如何将复制叉开始生成复制DNA的网络。了解蛋白质和蛋白质复合物的结构，这些蛋白质和蛋白质复合物催化了原核生物和真核染色体的复制，而病毒对于在复制叉时结合关键成分的药物的发展至关重要。这将有助于发展新的抗菌，真菌和病毒化合物。 公共卫生相关性了解蛋白质和蛋白质复合物的结构，这些蛋白质和蛋白质复合物催化了原核生物和真核染色体的复制和病毒对结合这些蛋白质和结构的新药的合理设计至关重要。这项研究采用高分辨率电子显微镜，在细菌，真菌，真核细胞和病毒中以复制起源和复制叉的蛋白质复合物提供独特而直接的可视化，以提供确定新药物靶标所需的信息。