Development of HTS Assays for Bacterial DNA Replication

细菌 DNA 复制 HTS 检测的开发

• 批准号: 7004575
• 负责人: CHARLES S MCHENRY
• 金额: $ 41.12万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 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/7004575
• 关键词: Bacillus subtilis; DNA directed DNA polymerase; DNA replication; DNA replication origin; Escherichia coli; antibacterial agents; bacterial DNA; biotechnology; cell line; chemical genetics; computer program /software; drug discovery /isolation; fluorescence resonance energy transfer; genetic techniques; gram negative bacteria; gram positive bacteria; high throughput technology; replicase; small molecule; technology /technique development

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（研究者提供）：细菌中的DNA复制是由特定的起源结合蛋白启动的，该蛋白募集了解旋酶组装蛋白和复制性解旋酶。一旦组装在DNA上的解旋酶为原始酶提供了一个相互作用位点，该酶是生成DNA合成的RNA引物的酶。复制性解旋酶在募集细胞复制酶，即DNA聚合酶III全酶中起作用，该酶具有在不解离的情况下合成整个染色体的加工性。尽管存在这种潜力，但大多数复制均遇到损坏，导致复制叉破坏。可以通过特殊起源独立复制重新启动设备来抵消这种损坏，该设备可以重新组装复制叉。总的来说，这些复制反应至少采用了20种不同的必需蛋白质。这些蛋白质靶标及其之间发生的基本相互作用为开发抗菌物提供了有吸引力的靶标，并且还将成为开发化学遗传方法以扰动各种相互作用和反应阶段的理想系统。我们建议开发可靠的HTS筛选测定法以及适当的特异性测定和截面，以使发现有可能发展为抗菌物质和DNA复制途径的阶跃特异性扰动者的小分子抑制剂。这些系统将使用模型克（ - ）和克（+）生物，大肠杆菌和枯草芽孢杆菌开发。这些生物与最常见的人类病原体密切相关，生物浓度类别A分别是生物体，鼠疫耶尔森氏菌和炭疽芽孢杆菌。