Control of DNA Topology

本项目主要研究IA型细菌DNA拓扑异构酶的作用机制， 利用这类细菌拓扑异构酶作为新抗生素的靶标。一个关键问题仍然是 IA型DNA拓扑异构酶涉及构象变化的机制， 分离与切割的DNA的两个不同末端结合的酶结构域， 可以发生在被切割的DNA链上。提高对IA型DNA拓扑异构酶的认识 酶的构象和底物的动态变化的信息是必需的 交互.结核分枝杆菌拓扑异构酶I的蛋白质中没有半胱氨酸残基 顺序定点诱变将用于在选择的位置引入独特的半胱氨酸残基。 用于目的1和2中的机理研究的酶结构中的位置。我们最近发现 在细菌IA型DNA拓扑异构酶的特定保守残基处的突变导致SOS 诱导和细菌细胞杀伤。这对这些酶作为抗菌剂具有非常重要的意义 目标3将继续跟进。 目标1.化学反应基团将被引入到蛋白质的特定位置，以绘制变化 在DNA-蛋白质相互作用中，酶在松弛机制的不同步骤中进行。 目标二。许多荧光探针将被放置在蛋白质结构的关键位置， 用于研究蛋白质构象的变化，这些变化对DNA通过所需的事件至关重要。 酶的作用将鉴定影响酶构象变化的突变。 目标3.在细菌IA型DNA拓扑异构酶的特定保守残基处的突变可导致 SOS诱导和细菌细胞杀伤将进一步分析。其他残基突变对 由鉴定的突变体形成的具有裂解DNA的共价中间体的稳定化将 研究，模拟酶的活性位点的扰动。 对所有常见抗生素具有耐药性的病原菌的出现代表了一个严峻的挑战， 公共卫生这项研究的结果有可能导致一类新的 抗生素

This project is focused on the mechanism of bacterial type IA DMAtopoisomerases and the potential of utilizing this class of bacterial topoisomerases as target for novel antibiotics. A key question that remains to be answered for type IA DNA topoisomerase concerns the mechanism of conformational change that separates the enzyme domains bound to the two different ends of the cleaved DNA, so that strand passage can take place across the cleaved DNA strand. To advance the knowledge on type IA DNA topoisomerase mechanism, information is needed on the dynamic changes in enzyme conformation and enzyme-substrate interactions. Mycobacterium tuberculosis topoisomerase I does not have any cysteine residue in its protein sequence. Site-directed mutagenesis will be used to introduce unique cysteine residues at selected positions in the enzyme structure for the mechanistic studies in Aims 1 and 2. We have recently discovered that mutation at a specific conserved residue of bacterial type IA DNA topoisomerases leads to SOS induction and bacterial cell killing. This has highly significant implication for these enzymes as antibacterial drug target and will be followed up in Aim 3. Aim 1. Chemically reactive groups will be introduced into specific positions of the protein to map the changes in DNA-protein interactions as the enzyme proceeds in the different steps of the relaxation mechanism. Aim 2. A number of fluorescence probes will be placed in strategic positions of the protein structure and utilized to study the changes in protein conformation critical for the DNA passage event required for the enzyme action. Mutations that affect enzyme conformational change will be identified. Aim 3. Mutations at a specific conserved residue of bacterial type IA DNA topoisomerases that can lead to SOS induction and bacterial cell killing will be further analyzed. The effect of mutations at other residues on the stabilization of the covalent intermediate with cleaved DNA formed by the identified mutant will be investigated, to model the resulting perturbation of the active site of the enzyme. The emergence of pathogenic bacteria resistant to all common antibiotics represent a critical challenge in public health. The results of this research has the potential to lead to the development of a novel class of antibiotics.