Bacterial cell killing by topoisomerase I mediated DNA lesion

拓扑异构酶 I 介导的 DNA 损伤杀死细菌细胞

• 批准号: 7333269
• 负责人: Yuk-Ching Tse-Dinh
• 金额: $ 37.15万
• 依托单位: NEW YORK MEDICAL COLLEGE
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-02-01 至 2011-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7333269
• 关键词: Anti-Bacterial Agents; Antibiotics; Bacteria; Bacterial DNA Topoisomerase I; Bacterial Typing; Biological Assay; Cells; Class; Cleaved cell; Collaborations; Community Hospitals; Complex; Condition; DNA; DNA Fingerprinting; DNA Topoisomerases; DNA biosynthesis; DNA lesion; Development; Emerging Communicable Diseases; Enzymes; Escherichia coli; Face; Future; General Population; Genes; Genetic Recombination; Genetic Transcription; Genomic Library; Goals; Gram-Negative Bacteria; Growth; In Vitro; Infection; Laboratories; Lead; Lesion; Libraries; Malignant Neoplasms; Measurement; Mediating; Modeling; Monitor; Mutagenesis; Mutation; Pathway interactions; Personal Satisfaction; Plasmids; Predisposition; Prevention; Protein Biosynthesis; Proteins; Public Health; Pulsed-Field Gel Electrophoresis; Recombinants; Research; Research Personnel; Resistance; Role; Screening procedure; Therapeutic Agents; Topoisomerase; Type I DNA Topoisomerases; Yersinia pestis; bactericide; biodefense; cell killing; cellular targeting; desire; follow-up; genetic strain; human TOP1 protein; killings; medical schools; mutant; novel; pathogen; pathogenic bacteria; programs; recombinational repair; response; small molecule

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.

DMA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，在裂解的DNA和II型或类型IB之间形成的共价复合物的捕获 治疗剂的DNA拓扑异构酶导致癌症或细菌细胞杀死。类型类 IA DNA拓扑异构酶是新型治疗剂的有希望的靶标，但是靶向Ia型的分子 尚未发现DNA拓扑异构酶。我们已经确定了耶尔森氏菌的突变体 拓扑异构酶I由于稳定而在大肠杆菌细胞中表达时可能导致大量杀戮 与裂解DNA的共价复合物。这是细菌细胞杀死的首次演示 由IA型DNA拓扑异构酶形成的累积共价复合物。该项目的具体目的是： 1。将在哈佛医学院的NSRB设施中使用高通道测定 可用于小分子的150,000种化合物，可导致共价复合物的积累 由重组Yersinia Pestis拓扑异构酶I形成。HITS将以体外拓扑异构酶为特征 测定和细菌细胞杀死，并与NSRB的药物化学家合作进一步开发。 2。研究稳定的IA型DNA拓扑异构酶共价稳定的细菌细胞杀死的机理 综合大肠杆菌表达突变体鼠疫托拓拓扑异构酶I，形成稳定的共价复合物 将被研究。在不同的生长下将比较对拓扑异构酶I诱导的诱导的敏感性的敏感性 确定DNA复制和蛋白质合成在细胞杀死机制中的作用的条件。 3。确定影响细菌对被困型iatopoyomerase杀死的可疑性的其他因素 将研究裂解的复合物，大肠杆菌菌株，并研究重组和修复途径中的突变。 E. 多拷贝质粒中的大肠带基因组文库将用于鉴定在较高的 水平可以赋予对拓扑异构酶I介导的细胞杀伤的耐药性。转座子诱变将被携带 筛选出具有提高敏感性或对细胞杀伤性的敏感性或抗性的突变体。 致病细菌对所有常见抗生素具有抗性的病原细菌是一个关键的挑战 公共卫生。使用细菌病原体的未来恐怖袭击可能涉及抗药性 当前的抗生素。这项研究有可能导致发现一种新型的抗生素。