Due to the prevalence of antibiotic-resistant bacteria, there is a need for the development of novel antibiotics. Topoisomerases control DNA topology via the breaking and rejoining of DNA backbone bonds coupled to DNA strand passage. Eukaryotic topoisomerases and DNA gyrase are known targets for anti-cancer and anti-bacterial drugs. Most of these drugs act by altering the cleavage-religation equilibrium of topoisomerases, resulting in accumulation of cleaved complexes in the cell. In bacteria, topoisomerase I modulates the level of DNA supercoiling along with DNA gyrase. Transcription of many bacterial genes, including genes required for virulence, has been shown to be sensitive to alteration of topoisomerase activities. There is little homology between bacterial topoisomerase I and its mammalian counterpart so it is attractive as a new target in the search for new antibiotics. This requires better understanding of the mechanism and regulation of bacterial topoisomerase I.
The Specific Aims proposed for this project include: 1. The mechanism of DNA cleavage-religation by E. coli DNA topoisomerase I will be investigated. A) Conserved amino acid residues found int he active site possibly involved in the binding of Mg(II) and cleavage religation will be altered by site-directed mutagenesis. The effect of such mutations on different enzymatic properties will be assessed. B) Oligonucleotide substrates with modification in the phosphodiester backbone bond will be utilized to test the substrate-assisted model of catalysis. 2. The regulation of topA transcription in E. coli will be studied to determine how the cell maintains or adjusts topoisomerase I level in response changes in growth environment. A) The effect of growth conditions on the in vivo expression from the individual topA promoters will be determined. B) Effect of mutations in the possible regulatory factors on the expression of individual promoters will be assessed. C) The effect of different E. coli sigma factors or transcription regulators on the in vitro transcription of the topA promoters with purified components will be characterized.
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