Bacterial cell killing topoisomerase I--DNA lesion

描述（申请人提供）：DNA拓扑异构酶是参与DNA复制、转录和重组的普遍存在的酶。众所周知，治疗剂捕获在切割DNA和II型或IB型DNA拓扑异构酶之间形成的共价复合物可导致癌症或细菌细胞的死亡。IA型DNA拓扑异构酶是一种很有前景的新型治疗药物靶点，但针对IA型DNA拓扑异构酶的分子尚未被发现。我们已经鉴定出鼠疫耶尔森氏菌拓扑异构酶I的突变体，当在大肠杆菌细胞中表达时，由于与切割DNA的共价复合物的稳定性，可以导致广泛的杀伤。这是第一次证明细菌细胞由IA型DNA拓扑异构酶形成的积累共价复合物杀死。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15万种可用的小分子化合物，这些化合物将导致重组鼠疫耶尔森菌拓扑异构酶i形成的共价复合物的积累。命中将通过体外拓扑异构酶测定和细菌细胞杀伤进行表征，并与NSRB的药物化学家合作进一步开发。2. 为了研究稳定型IA型DNA拓扑异构酶共价复合物杀死细菌细胞的机制，将对表达突变型鼠疫菌拓扑异构酶I形成稳定共价复合物的大肠杆菌进行研究。将比较不同生长条件下对拓扑异构酶I诱导的DNA损伤的敏感性，以确定DNA复制和蛋白质合成在细胞杀伤机制中的作用。3. 为了确定影响细菌被捕获型IA拓扑异构酶切割复合体杀死敏感性的其他因素，将研究重组和修复途径突变的大肠杆菌菌株。多拷贝质粒中的大肠杆菌基因组文库将用于鉴定当以更高水平表达时可以赋予对拓扑异构酶I介导的细胞杀伤的抗性的蛋白质。转座子诱变将用于筛选对细胞杀伤具有更高敏感性或抗性的突变体。

DESCRIPTION (provided by applicant): DNA 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 therapeutic 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 accumulated 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 stabilized covalent complex will be studied. Sensitivity to topoisomerase I induced DNA lesion 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 susceptibility of bacteria to killing by trapped type IA topoisomerase 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 represents 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.