Control of DNA Topology

描述（由申请人提供）：本项目主要研究细菌IA型DMA拓扑异构酶的作用机制，以及利用这类细菌拓扑异构酶作为新型抗生素靶点的潜力。对于IA型DNA拓扑异构酶，仍有一个关键问题有待解答，即分离与断裂DNA的两个不同末端结合的酶结构域的构象变化机制，以便在断裂的DNA链上进行链传递。为了进一步了解IA型DNA拓扑异构酶的机制，需要了解酶的构象和酶-底物相互作用的动态变化。结核分枝杆菌拓扑异构酶I在其蛋白序列中不含半胱氨酸残基。位点定向诱变将用于在酶结构的选定位置引入独特的半胱氨酸残基，用于Aims 1和2的机制研究。我们最近发现，细菌IA型DNA拓扑异构酶的特定保守残基突变导致SOS诱导和细菌细胞死亡。这对这些酶作为抗菌药物靶点具有重要意义，将在Aim 3中进行后续研究。

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.