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.
|Zhou, Qingxuan; Gomez Hernandez, Mario E; Fernandez-Lima, Francisco et al. (2018) Biochemical Basis of E. coli Topoisomerase I Relaxation Activity Reduction by Nonenzymatic Lysine Acetylation. Int J Mol Sci 19:|
|Banda, Srikanth; Cao, Nan; Tse-Dinh, Yuk-Ching (2017) Distinct Mechanism Evolved for Mycobacterial RNA Polymerase and Topoisomerase I Protein-Protein Interaction. J Mol Biol 429:2931-2942|
|Zhou, Qingxuan; Zhou, Yan Ning; Jin, Ding Jun et al. (2017) Deacetylation of topoisomerase I is an important physiological function of E. coli CobB. Nucleic Acids Res 45:5349-5358|
|Cheng, Bokun; Zhou, Qingxuan; Weng, Liwei et al. (2017) Identification of proximal sites for unwound DNA substrate in Escherichia coli topoisomerase I with oxidative crosslinking. FEBS Lett 591:28-38|
|Tan, Kemin; Cao, Nan; Cheng, Bokun et al. (2016) Insights from the Structure of Mycobacterium tuberculosis Topoisomerase I with a Novel Protein Fold. J Mol Biol 428:182-193|
|Tiwari, Purushottam B; Chapagain, Prem P; Banda, Srikanth et al. (2016) Characterization of molecular interactions between Escherichia coli RNA polymerase and topoisomerase I by molecular simulations. FEBS Lett 590:2844-51|
|Banda, Srikanth; Tiwari, Purushottam Babu; Darici, Yesim et al. (2016) Investigating direct interaction between Escherichia coli topoisomerase I and RecA. Gene 585:65-70|
|Tan, Kemin; Zhou, Qingxuan; Cheng, Bokun et al. (2015) Structural basis for suppression of hypernegative DNA supercoiling by E. coli topoisomerase I. Nucleic Acids Res 43:11031-46|
|Cheng, Bokun; Annamalai, Thirunavukkarasu; Sandhaus, Shayna et al. (2015) Inhibition of Zn(II) binding type IA topoisomerases by organomercury compounds and Hg(II). PLoS One 10:e0120022|
|Tse-Dinh, Yuk-Ching (2015) Targeting bacterial topoisomerase I to meet the challenge of finding new antibiotics. Future Med Chem 7:459-71|
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