Development of HTS Assays for Bacterial DNA Replication

描述（由研究者提供）：细菌中的DNA复制由一种特异性的起始结合蛋白启动，该蛋白募集解旋酶组装蛋白和复制性解旋酶。解旋酶一旦组装在DNA上，就会为引发酶提供相互作用位点，引发酶是一种产生DNA合成RNA引物的酶。复制解旋酶也在募集细胞复制酶中起作用，DNA聚合酶III全酶具有合成整个染色体而不解离的持续合成能力。尽管有这种潜力，大多数复制酶遇到的损害，导致复制叉破坏。这种损害可以抵消一个特殊的起源独立的复制重新启动装置，可以重新组装复制叉。总之，这些复制反应使用至少20种不同的必需蛋白质。这些蛋白质靶标和它们之间发生的基本相互作用为抗菌剂的开发提供了有吸引力的靶标，并且还将作为开发化学遗传方法以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筛选试验以及适当的特异性试验和counterscreens，使小分子抑制剂，有可能被开发成抗菌药物和DNA复制途径的步骤特异性干扰剂的发现。这些系统将使用模型革兰氏（-）和革兰氏（+）微生物，E。coli和B.枯草杆菌。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.