Development of HTS Assays for Bacterial DNA Replication

描述（由研究者提供）：细菌中的 DNA 复制是由特定的起点结合蛋白启动的，该蛋白招募解旋酶组装蛋白和复制解旋酶。解旋酶一旦组装到 DNA 上，就会为引物酶提供相互作用位点，引物酶为 DNA 合成生成 RNA 引物。复制解旋酶还在招募细胞复制酶（DNA 聚合酶 III 全酶）方面发挥作用，该酶具有合成整个染色体而不解离的持续能力。尽管有这种潜力，大多数复制酶都会受到损坏，导致复制叉被破坏。这种损坏可以通过一种特殊的独立于起源的复制重启装置来抵消，该装置可以重新组装复制叉。总的来说，这些复制反应至少使用 20 种不同的必需蛋白质。这些蛋白质靶标以及它们之间发生的重要相互作用为抗菌药物的开发提供了有吸引力的靶标，并且也将作为开发化学遗传方法以扰乱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物和 DNA 复制途径的步骤特异性干扰剂的小分子抑制剂。这些系统将使用模型革兰氏 (-) 和革兰氏 ( ) 生物体、大肠杆菌和枯草芽孢杆菌进行开发。这些生物体分别与最常见的人类病原体和生物防御 A 类生物体、鼠疫耶尔森氏菌和炭疽杆菌密切相关。

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.