CONTROL OF DNA TOPOLOGY

由于抗药性细菌的流行，有必要 新型抗生素的开发。拓扑异构酶控制DNA拓扑 通过断裂和重新连接连接到DNA链上的DNA主链 通过。真核细胞的拓扑异构酶和DNA旋转酶是已知的靶标 抗癌和抗菌药物。这些药物中的大多数是通过改变 拓扑异构酶的裂解-宗教平衡，导致 裂解的复合体在细胞内积累。在细菌中，拓扑异构酶 我与DNA旋转酶一起调节DNA超螺旋的水平。 许多细菌基因的转录，包括 毒力已被证明对拓扑异构酶的变化敏感 活动。细菌拓扑异构酶I和细菌拓扑异构酶I之间几乎没有同源性 它是哺乳动物的同类，所以它作为搜索的新目标很有吸引力 寻找新的抗生素。这需要更好地理解这种机制。 和细菌拓扑异构酶I的调控。 这项计划建议的具体目标包括： 1.大肠杆菌DNA拓扑异构酶切割DNA的机制 我会被调查的。 A)活性中心可能存在保守的氨基酸残基 参与镁离子结合的裂解宗教将发生改变 通过定点突变。这些突变对不同物种的影响 将对酶的性质进行评估。 B)磷酸二酯修饰的寡核苷酸底物 主链将被用来测试衬底辅助模型 催化作用。 2.研究topA在大肠杆菌中的转录调控 确定细胞如何维持或调节拓扑异构酶I的水平 应对生长环境的变化。 A)生长条件对体内表达的影响 将确定单独的topA启动子。 B)可能的调节因子突变对 将对单个启动子的表达进行评估。 C)不同的大肠杆菌西格玛因子或转录的影响 对topA启动子体外转录的调节作用 将对提纯的成分进行表征。

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.