Development of HTS Assays for Bacterial DNA Replication

细菌 DNA 复制 HTS 检测的开发

• 批准号: 7347431
• 负责人: CHARLES S MCHENRY
• 金额: $ 32.52万
• 依托单位: UNIVERSITY OF COLORADO
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-02-01 至 2008-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7347431
• 关键词: Animal Model; Anti-Bacterial Agents; Bacillus anthracis; Bacteria; Binding Proteins; Biochemical; Biological Assay; Categories; Cells; Chromosomes; DNA; DNA Polymerase III; DNA Primase; DNA biosynthesis; DNA chemical synthesis; DNA-Directed DNA Polymerase; Development; Dissociation; Double Strand Break Repair; Enzymes; Escherichia coli; Exhibits; Genetic; Goals; Holoenzymes; Human; Modeling; Molecular Target; Organism; Pathway interactions; Play; Proteins; RNA primers; Reaction; Reagent; Recruitment Activity; Replication Initiation; Replicon; Research Personnel; Resistance development; Restart; Role; Screening procedure; Site; Specificity; Staging; System; Technology; Yersinia pestis; antimicrobial; biodefense; chemical genetics; helicase; high throughput screening; inhibitor/antagonist; novel; pathogen; prototype; replicase; small molecule

DESCRIPTION (provided by investigator): DNA replication, in bacteria, is initiated by a specific origin binding protein that recruits helicase assembly proteins and the replicative helicase. The helicase, once assembled on DNA, provides an interaction site for primase, the enzyme that generates RNA primers for DNA synthesis. The replicative helicase also plays a role in recruiting the cellular replicase, the DNA polymerase III holoenzyme that has the processivity to synthesize the entire chromosome without dissociation. In spite of this potential, most replicases encounter damage, resulting in replication fork destruction. This damage can be counteracted by a special origin independent replication restart apparatus that can reassemble replication forks. Altogether, these replicative reactions employ at least 20 different essential proteins. These protein targets and the essential interactions that occur between them provide attractive targets for the development of antibacterials, and will also serve as an ideal system for developing chemical genetic approaches to perturb the various interactions and reaction stages. We propose to develop robust HTS screening assays as well as appropriate specificity assays and counterscreens to enable the discovery of small molecule inhibitors that have the potential to be developed into antibacterials and step-specific perturbants of DNA replication pathways. These systems will be developed using model Gram (-) and Gram (+) organisms, E. coli and B. subtilis. These organisms are closely related to most common human pathogens and the biodefense category A organisms, Yersinia pestis and Bacillus anthracis, respectively.

描述（由研究者提供）：在细菌中，DNA复制是由一个特定的起始结合蛋白发起的，该蛋白招募解旋酶组装蛋白和复制解旋酶。解旋酶一旦组装在DNA上，就为引物酶提供了一个相互作用位点，引物酶产生用于DNA合成的RNA引物。复制解旋酶还在募集细胞复制酶中发挥作用，细胞复制酶是一种DNA聚合酶III全酶，具有合成整个染色体而不解离的能力。尽管有这种潜力，但大多数复制酶都会受到损害，导致复制分叉破坏。这种损害可以通过一种特殊的独立于起源的复制重启装置来抵消，这种装置可以重新组装复制分叉。总的来说，这些复制反应使用了至少20种不同的基本蛋白质。这些蛋白质靶点和它们之间发生的基本相互作用为抗菌药物的开发提供了有吸引力的靶点，也将作为开发化学遗传方法来干扰各种相互作用和反应阶段的理想系统。我们建议开发强大的HTS筛选分析以及适当的特异性分析和反筛选，以发现有潜力开发成抗菌药和DNA复制途径的步骤特异性干扰物的小分子抑制剂。这些系统将使用模型革兰氏（-）和革兰氏（+）有机体，大肠杆菌和枯草芽孢杆菌来开发。这些生物分别与大多数常见的人类病原体和生物防御A类生物，鼠疫耶尔森菌和炭疽芽孢杆菌密切相关。