Development of HTS Assays for Bacterial DNA Replication

描述(由研究人员提供)：在细菌中，DNA复制是由一种特定的起始结合蛋白启动的，该结合蛋白招募解旋酶组装蛋白和复制的解旋酶。解旋酶一旦组装到DNA上，就为底物酶提供了一个相互作用的部位，底物酶是一种为DNA合成产生RNA引物的酶。复制解旋酶还在招募细胞复制酶方面发挥作用，细胞复制酶是DNA聚合酶III的全酶，具有不解离地合成整个染色体的过程。尽管存在这种可能性，大多数复制都会遭到破坏，从而导致复制分叉的破坏。这种损害可以通过一种特殊的与起始点无关的复制重启设备来抵消，该设备可以重新组装复制叉子。总而言之，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，也将成为开发扰乱各种相互作用和反应阶段的化学遗传方法的理想系统。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，以使发现小分子抑制剂具有开发成抗菌药物和DNA复制途径的步骤特异性扰动的潜力。这些系统将使用模式革兰氏(-)和革兰氏(-)生物，大肠杆菌和枯草杆菌来开发。这些生物分别与最常见的人类病原体和A类生物防御生物--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.