CONTROL OF DNA TOPOLOGY

由于抗生素抗性细菌的流行，需要 新型抗生素的开发。 拓扑异构酶控制DNA拓扑结构 通过与DNA链偶联的DNA骨架键的断裂和重新连接 个通道好吗 真核拓扑异构酶和DNA促旋酶是已知的 抗癌和抗菌药物。 大多数药物通过改变 拓扑异构酶的裂解-再连接平衡，导致 裂解的复合物在细胞中的积累。 在细菌中，拓扑异构酶 I与DNA促旋酶一起调节DNA超螺旋沿着的水平。 许多细菌基因的转录，包括 毒力，已被证明是敏感的改变拓扑异构酶 活动 细菌拓扑异构酶I和 它的哺乳动物对应物，因此它是有吸引力的搜索新目标 新的抗生素。 这就需要更好地理解这一机制 和细菌拓扑异构酶I的调节。 为该项目提出的具体目标包括： 1. 探讨了E.大肠杆菌DNA拓扑异构酶 我会被调查的 A）在活性位点中发现的保守氨基酸残基可能 参与Mg（II）结合和裂解再连接的 通过定点突变。 这种突变对不同的 将评估酶性质。 B）在磷酸二酯中具有修饰的寡核苷酸底物 骨干债券将被用来测试基板辅助模型， 催化作用 2. topA基因在E.将研究大肠杆菌， 确定细胞如何维持或调节拓扑异构酶I水平， 适应生长环境的变化。 A）生长条件对来自大肠杆菌的体内表达的影响 将确定单个topA启动子。 B）可能的调节因子中的突变对细胞的影响。 评估单个启动子的表达。 C）不同E.大肠杆菌σ因子或转录 调节因子对topA启动子的体外转录的影响， 将表征纯化的组分。

Due to the prevalence of antibiotic-resistant bacteria, there is a need for the development of novel antibiotics. Topoisomerases control DNA topology via the breaking and rejoining of DNA backbone bonds coupled to DNA strand passage. Eukaryotic topoisomerases and DNA gyrase are known targets for anti-cancer and anti-bacterial drugs. Most of these drugs act by altering the cleavage-religation equilibrium of topoisomerases, resulting in accumulation of cleaved complexes in the cell. In bacteria, topoisomerase I modulates the level of DNA supercoiling along with DNA gyrase. Transcription of many bacterial genes, including genes required for virulence, has been shown to be sensitive to alteration of topoisomerase activities. There is little homology between bacterial topoisomerase I and its mammalian counterpart so it is attractive as a new target in the search for new antibiotics. This requires better understanding of the mechanism and regulation of bacterial topoisomerase I. The Specific Aims proposed for this project include: 1. The mechanism of DNA cleavage-religation by E. coli DNA topoisomerase I will be investigated. A) Conserved amino acid residues found int he active site possibly involved in the binding of Mg(II) and cleavage religation will be altered by site-directed mutagenesis. The effect of such mutations on different enzymatic properties will be assessed. B) Oligonucleotide substrates with modification in the phosphodiester backbone bond will be utilized to test the substrate-assisted model of catalysis. 2. The regulation of topA transcription in E. coli will be studied to determine how the cell maintains or adjusts topoisomerase I level in response changes in growth environment. A) The effect of growth conditions on the in vivo expression from the individual topA promoters will be determined. B) Effect of mutations in the possible regulatory factors on the expression of individual promoters will be assessed. C) The effect of different E. coli sigma factors or transcription regulators on the in vitro transcription of the topA promoters with purified components will be characterized.