Bacterial cell killing by topoisomerase I mediated DNA lesion

DNA拓扑异构酶是广泛存在的参与DNA复制、转录和重组的酶。 众所周知，切割的DNA与II型或IB型之间形成的共价复合物的捕获是通过将DNA与II型或IB型之间形成的共价复合物结合而实现的。 治疗剂的DNA拓扑异构酶导致杀死癌症或细菌细胞。类型的类 IA型DNA拓扑异构酶是一个很有前途的新的治疗药物的靶点，但针对IA型的分子， DNA拓扑异构酶尚未发现。我们发现了一种鼠疫菌的突变体 拓扑异构酶I，当在E.大肠杆菌细胞由于稳定的 与切割的DNA的共价复合物。这是第一次证明细菌细胞杀死从 由IA型DNA拓扑异构酶形成的交联共价复合物。该项目的具体目标是： 1.哈佛医学院的NSRB设施将使用高通量测定来筛选 150，000种化合物可用于导致共价复合物积累的小分子 由重组鼠疫杆菌拓扑异构酶I形成。命中将通过体外拓扑异构酶来表征 检测和细菌细胞杀伤，并与NSRB的药物化学家合作进一步开发。 2.目的：探讨IA型拓扑异构酶共价键稳定化对细菌细胞的杀伤机制 complex，E. coli中表达突变体Y.形成稳定的共价复合物的鼠疫拓扑异构酶I 将被研究。比较不同生长条件下对拓扑异构酶I诱导的DNA酶切的敏感性 条件来确定DNA复制和蛋白质合成在细胞杀伤机制中的作用。 3.目的：探讨其他影响诱捕型IA拓扑异构酶杀灭细菌能力的因素 切割复合体，E.将研究在重组和修复途径中具有突变的大肠杆菌菌株。大肠 多拷贝质粒中的大肠杆菌基因组文库将用于鉴定当在较高表达水平表达时的蛋白质。 水平，可以赋予对拓扑异构酶I介导的细胞杀伤的抗性。将进行转座子诱变 以筛选对细胞杀伤敏感性或抗性增加的突变体。 对所有常见抗生素具有耐药性的病原菌的出现代表了一个严峻的挑战， 公共卫生未来使用细菌病原体的恐怖袭击可能涉及对 当前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.