Control of DNA Topology

描述（由申请人提供）：本项目的重点是IA型细菌DMA拓扑异构酶的机制以及利用此类细菌拓扑异构酶作为新型抗生素靶标的潜力。IA型DNA拓扑异构酶的一个关键问题是构象变化的机制，该机制将结合在切割的DNA的两个不同末端的酶结构域分开，使得链通过可以跨越切割的DNA链发生。为了进一步了解IA型DNA拓扑异构酶的作用机制，需要了解酶的构象动态变化和酶与底物的相互作用。结核分枝杆菌拓扑异构酶I在其蛋白质序列中没有任何半胱氨酸残基。将使用定点诱变在酶结构中的选定位置引入独特的半胱氨酸残基，用于目的1和2中的机制研究。我们最近发现，在一个特定的保守残基的IA型细菌DNA拓扑异构酶的突变导致SOS诱导和细菌细胞杀伤。这对于这些酶作为抗菌药物靶标具有高度重要的意义，并将在目标3中进行跟踪。 目标1.化学反应基团将被引入蛋白质的特定位置，以绘制DNA-蛋白质相互作用的变化，因为酶在松弛机制的不同步骤中进行。 目标2.许多荧光探针将被放置在蛋白质结构的关键位置，并用于研究蛋白质构象的变化，这些变化对于酶作用所需的DNA通过事件至关重要。将鉴定影响酶构象变化的突变。 目标3。将进一步分析可导致SOS诱导和细菌细胞杀伤的IA型细菌DNA拓扑异构酶的特定保守残基处的突变。将研究在其他残基处的突变对共价中间体与由鉴定的突变体形成的裂解DNA的稳定性的影响，以模拟所产生的酶活性位点的扰动。 对所有常见抗生素产生耐药性的病原菌的出现是公共卫生领域的一个重大挑战。这项研究的结果有可能导致一类新型抗生素的开发。

DESCRIPTION (provided by applicant): This project is focused on the mechanism of bacterial type IA DMA topoisomerases 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.