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，因此迫切需要开发针对新靶点的新抗生素。 细菌拓扑异构酶I是一个有前途的新靶标，用于用对金黄色葡萄球菌具有4.0 μ g MIC的先导化合物进行抗菌治疗。 E.大肠杆菌拓扑异构酶I是细菌拓扑异构酶I的最佳研究实例，并且与来自革兰氏阳性和革兰氏阴性细菌的拓扑异构酶I共享广泛的同源性。 以拓扑异构酶I为靶点的抑制DNA再连接的药物会导致细胞杀伤，其机制与许多以细菌DNA促旋酶和人类拓扑异构酶为靶点的药物相似。 拓扑异构酶I功能的丧失也可能影响细菌响应发病机制中遇到的环境挑战的能力。 本项目的长期目标是阐明E.大肠杆菌拓扑异构酶I，这将大大有助于开发针对这类酶的新型细菌制剂。 该提案的目标包括：1.通过不同的诱变方法进行结构-功能分析，以确定E. coli拓扑异构酶I 2.在不存在和存在DNA的情况下，拓扑异构酶I的有限蛋白水解和化学切割，以鉴定由于酶构象变化或DNA底物保护而改变的切割位点。 3.通过淘选鉴定为拓扑异构酶I4的潜在抑制剂的肽序列的测试。拓扑异构酶I参与细菌适应生存环境挑战的分子基础研究。

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.