Bacterial cell killing by topoisomerase I mediated DNA lesion

DNA拓扑异构酶是DNA复制、转录和重组过程中普遍存在的酶。 众所周知，捕获切割的DNA和II型或IB型之间形成的共价络合物 治疗药物的DNA拓扑异构酶可以杀死癌细胞或细菌细胞。类型的类别 IA DNA拓扑异构酶是一种很有前途的新型治疗药物靶点，但分子靶向的是IA型 DNA拓扑异构酶尚未被发现。我们已经鉴定出一株鼠疫耶尔森氏菌的突变体 拓扑异构酶I在大肠杆菌中表达时，由于其稳定性，可导致广泛的杀伤 与切割的DNA形成的共价复合体。这是第一次演示细菌细胞从 由IA型DNA拓扑异构酶形成的累积共价复合体。该项目的具体目标是： 1.将在哈佛医学院的NSRB设施中使用高通量测试来筛选 可供小分子使用的150,000种化合物将导致共价络合物的积累 重组鼠疫菌拓扑异构酶I形成HITS体外拓扑异构酶的特征 测试和细菌细胞杀灭，并与NSRB的药物化学家合作进一步开发。 2.探讨稳定型IA DNA拓扑异构酶共价杀灭细菌细胞的机制 表达突变的鼠疫杆菌拓扑异构酶I，形成稳定的共价复合体 将会被研究。将比较不同生长条件下对拓扑异构酶I引起DNA损伤的敏感性 确定DNA复制和蛋白质合成在细胞杀伤机制中的作用条件。 3.确定影响捕捉型IAS杀灭细菌易感性的其他因素 裂解复合体，具有重组和修复途径突变的大肠杆菌菌株将被研究。一个E。 大肠杆菌基因组文库中的多拷贝质粒将被用来鉴定在较高水平表达的蛋白质 水平，可对拓扑异构酶I介导的细胞杀伤产生抵抗。将进行转座子诱变 以筛选对细胞杀伤具有更高敏感性或抵抗力的突变体。 对所有常见抗生素耐药的病原菌的出现是对 公共卫生。未来使用细菌病原体的恐怖袭击可能涉及到对 目前的抗生素。这项研究有可能导致发现一类新的抗生素。

DMA topoisomerases are ubiquitous enzymes involved in DNA replication, transcription and recombination. It is well known that trapping of covalent complexes formed between cleaved DNA and type II or type IB DNA topoisomerases by therapeutic agents leads to the killing of cancer or bacterial cells. The class of type IA DNA topoisomerases is a promising target for novel therapetuic agents, but molecules targeting type IA DNA topoisomerases have not been discovered. We have identified a mutant of Yersinia pestis topoisomerase I that can result in extensive killing when expressed in E. coli cells due to the stabilization of the covalent complex with cleaved DNA. This is the first demonstration of bacterial cell killing from accumlated covalent complex formed by a type IA DNA topoisomerase. The specific aims of the project are: 1. A high-through-put assay will be utilized at the NSRB facility at Harvard Medical School to screen the 150,000 compounds available for small molecules that will result in accumulation of covalent complex formed by recombinant Yersinia pestis topoisomerase I. Hits will be characterized by in vitro topoisomerase assay and bacterial cell killing and further developed in collaboration with medicinal chemists at NSRB. 2. To investigate the mechanism of bacterial cell killing by stabilized type IA DNA topoisomerase covalent complex, E. coli expressing the mutant Y. pestis topoisomerase I that forms the stablized covalent complex will be studied. Sensitivity to topoisomerase I induced DNAlesion will be compared under different growth conditions to determine the role of DNA replication and protein synthesis in the cell killing mechanism. 3. To identify other factors influencing the suscepbility of bacteria to killing by trapped type IAtopoisomerase cleaved complex, E. coli strains with mutations in recombination and repair pathways will be studied. An E. coli genomic library in a multi-copy plasmid will be used to identify proteins that when expressed in a higher level, can confer resistance to topoisomerase I mediated cell killing. Transposon mutagenesis will be carried out to screen for mutants with increased sensitivity or resistance to the cell killing. The emergence of pathogenic bacteria resistant to all common antibiotics represent a critical challenge in public health. Future terrorist attacks employing bacterial pathogens could involve agents resistant to current antibiotics. This research has the potential to lead to the discovery of a novel class of antibiotics.