CONTROL OF DNA TOPOLOGY

由于最近出现了对目前使用的所有抗生素都具有耐药性的病原菌，因此迫切需要针对新的靶点开发新的抗生素。细菌拓扑异构酶I对金黄色葡萄球菌的MIC值为4.0杯，有望成为抗菌治疗的新靶点。大肠杆菌拓扑异构酶I是细菌拓扑异构酶I研究得最好的例子，它与革兰氏阳性和革兰氏阴性细菌的拓扑异构酶I有广泛的同源性。针对抑制DNA宗教的药物的拓扑异构酶I将导致细胞死亡，其机制类似于许多针对细菌DNA旋转酶和人类拓扑异构酶的药物。拓扑异构酶I功能的丧失也可能影响细菌对致病过程中遇到的环境挑战的反应能力。该项目的长期目标包括阐明大肠杆菌拓扑异构酶I的机制、调节和生理作用，这将极大地帮助开发针对这类酶的新型细菌制剂。这一建议的目的包括：1.通过不同的诱变方法进行结构-功能分析，以确定大肠杆菌拓扑异构酶I催化各个步骤所需的残基。2.在没有DNA和存在DNA的情况下，拓扑异构酶I的有限蛋白分解和化学裂解，以确定由于酶构象变化或DNA底物保护而改变的裂解位点。3.经PANING鉴定为拓扑异构酶I潜在抑制剂的多肽序列的测试4.拓扑异构酶I参与细菌适应生存环境挑战的分子基础的研究。

Due to the recent emergence of pathogenic bacteria resistant to all antibiotics currently used, there is an urgent lead to develop new antibiotics against novel targets. Bacterial topoisomerase I is a promising new target for antibacterial therapy with lead compounds having MIC's of 4.0 mug against Staphylococcus aureus. E. coli topoisomerase I is the best studied example of bacterial topoisomerase I and share extensive homology with topoisomerase I from both gram-positive and gram- negative bacteria. Topoisomerase I targeting drugs that inhibit DNA religation would lead to cell killing in a mechanism similar to those of many drugs targeting bacterial DNA gyrase and human topoisomerases. Loss of topoisomerase I function may also affect the ability of the bacteria to respond to environmental challenges encountered in pathogenesis. The long term goals of this project include the elucidation of the mechanism, regulation and physiological roles of E. coli topoisomerase I, which would greatly aid the development of novel bacterial agents targeting this class of enzyme. The aims for this proposal include: 1. Structure-function analysis by different mutagenesis approaches to identify residues required for the individual steps of catalysis by E. coli topoisomerase I 2. Limited proteolysis and chemical cleavage of topoisomerase I in the absence and presence of DNA to identify sites of cleavage altered due to either enzyme conformational change or protection by DNA substrate. 3. Test of peptide sequences identified by panning as potential inhibitor of topoisomerase I 4. Study of the molecular basis of topoisomerase I involvement in bacterial adaptation to environmental challenges for survival.