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致病菌，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是一种很有前景的新靶点，其先导化合物的MIC值为4.0。大肠杆菌拓扑异构酶I是研究得最好的细菌拓扑异构酶I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具有广泛的同源性。以拓扑异构酶I为靶点的药物抑制DNA重组，其机制与许多以细菌DNA回转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在发病过程中对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、调控和生理作用，这将极大地帮助开发针对这类酶的新型细菌制剂。本提案的目标包括：1。利用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i2催化各步骤所需的残基。在没有和存在DNA的情况下，拓扑异构酶I进行有限的蛋白质水解和化学裂解，以识别由于酶构象改变或DNA底物保护而改变的裂解位点。3. 经筛选鉴定为拓扑异构酶i4潜在抑制剂的肽序列的试验。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.