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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM054226-03
Application #
2685109
Study Section
Physiological Chemistry Study Section (PC)
Project Start
1996-04-01
Project End
2000-03-31
Budget Start
1998-04-01
Budget End
1999-03-31
Support Year
3
Fiscal Year
1998
Total Cost
Indirect Cost
Name
New York Medical College
Department
Biochemistry
Type
Schools of Medicine
DUNS #
City
Valhalla
State
NY
Country
United States
Zip Code
10595
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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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