Due to the recent emergence of pathogenic bacteria resistant to all antibiotics currently used, there is an urgent lead to develop new antibiotics against novel targets. Bacterial topoisomerase I is a promising new target for antibacterial therapy with lead compounds having MIC's of 4.0 mug against Staphylococcus aureus. E. coli topoisomerase I is the best studied example of bacterial topoisomerase I and share extensive homology with topoisomerase I from both gram-positive and gram- negative bacteria. Topoisomerase I targeting drugs that inhibit DNA religation would lead to cell killing in a mechanism similar to those of many drugs targeting bacterial DNA gyrase and human topoisomerases. Loss of topoisomerase I function may also affect the ability of the bacteria to respond to environmental challenges encountered in pathogenesis. The long term goals of this project include the elucidation of the mechanism, regulation and physiological roles of E. coli topoisomerase I, which would greatly aid the development of novel bacterial agents targeting this class of enzyme.
The aims for this proposal include: 1. Structure-function analysis by different mutagenesis approaches to identify residues required for the individual steps of catalysis by E. coli topoisomerase I 2. Limited proteolysis and chemical cleavage of topoisomerase I in the absence and presence of DNA to identify sites of cleavage altered due to either enzyme conformational change or protection by DNA substrate. 3. Test of peptide sequences identified by panning as potential inhibitor of topoisomerase I 4. Study of the molecular basis of topoisomerase I involvement in bacterial adaptation to environmental challenges for survival.
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