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.
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