The long term goals of this project are to understand how the activity, regulation and interactions of DNA topoisomerases control DNA topology and affect vital cellular functions. Drugs that initiate cell killing by trapping the covalent cleavage complex formed by type IB and type IIA topoisomerases are useful anti-cancer and anti-bacterial drugs. Previous results have shown that accumulation of type IA topoisomerase I cleavage complex can trigger bacterial cell death but specific inhibitors of type IA bacterial topoisomerase I remain to be discovered. The proposed research activities for the next funding period are relevant for both overcoming the critical barrier in the understanding of the mechanism of type IA topoisomerases and discovery of novel drugs targeting this class of topoisomerases. Topoisomerase I catalyzes the relaxation of DNA by cleaving a single strand in duplex DNA and passing the complementary strand through the break before religation of the cleaved strand. The molecular basis for the presence of a C nucleotide at the -4 position relative to the identified sites of cleavage by topoisomerase I has been revealed in the recently obtained crystal structure of the covalent cleavage complex. Site-directed mutagenesis and biochemical analysis will be used to test the hypothesis that interactions with this C nucleotide are important for relaxation of supercoiling catalyzed by E. coli DNA topoisomerase I. The results will reveal if the conservation of the C-nucleotide recognition during evolution is linked to the efficiency of the DNA winding activity required for physiological function. The molecular mechanism of how the passing DNA strand is guided by the enzyme during strand passage remains the critical barrier for progress in the understanding the overall enzyme catalytic mechanism. Partial DNA duplex molecules that are cleaved by E. coli topoisomerase I at a single site on one strand will be used for as substrates for determination of crystal structure of the full length enzyme as well as for mapping of site-specific protein-DNA interactions on the passing strand. The results will provide the molecular basis of how bacterial topoisomerase I catalyzes the important function of removal of excess negative supercoils by passing the complementary strand through the break after initial cleavage of a single strand. Genetic studies will test the hypothesis that certain perturbations of the non-covalent protein-DNA interactions observed in the crystal structure to be responsible for holding the 3'-OH portion of the cleaved DNA substrate in position for DNA religation could result in accumulation of DNA cleavage intermediate, leading to not only loss of topoisomerase I relaxation activity, but also potentially triggering the bacterial cell death pathway. Mutations in recombinant E. coli or Y. pestis topoisomerase I that can result in such perturbations of the non-covalent interactions with DNA will be identified by genetic selection for dominant lethal effects in E. coli and characterized biochemically. Success in these experiments would provide very useful information for discovery of novel antibacterial drugs targeting topoisomerase I specifically.

Public Health Relevance

This project studies the structure and mechanism of bacterial topoisomerase I relevant for discovery of specific inhibitors. The proposed work would greatly aide the development of new antibacterial compounds targeting topoisomerase I as part of the much needed arsenal to combat the global health problem of multi-drug drug resistant bacterial pathogens.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM054226-13A1
Application #
8237217
Study Section
Prokaryotic Cell and Molecular Biology Study Section (PCMB)
Program Officer
Reddy, Michael K
Project Start
1996-04-01
Project End
2012-12-31
Budget Start
2012-01-01
Budget End
2012-12-31
Support Year
13
Fiscal Year
2012
Total Cost
$313,950
Indirect Cost
$118,950
Name
New York Medical College
Department
Biochemistry
Type
Schools of Medicine
DUNS #
041907486
City
Valhalla
State
NY
Country
United States
Zip Code
10595
Tiwari, Purushottam Babu; Annamalai, Thirunavukkarasu; Cheng, Bokun et al. (2014) A surface plasmon resonance study of the intermolecular interaction between Escherichia coli topoisomerase I and pBAD/Thio supercoiled plasmid DNA. Biochem Biophys Res Commun 445:445-50
Sissi, Claudia; Cheng, Bokun; Lombardo, Valentina et al. (2013) Metal ion and inter-domain interactions as functional networks in E. coli topoisomerase I. Gene 524:253-60
Narula, Gagandeep; Annamalai, Thirunavukkarasu; Aedo, Sandra et al. (2011) The strictly conserved Arg-321 residue in the active site of Escherichia coli topoisomerase I plays a critical role in DNA rejoining. J Biol Chem 286:18673-80
Zhang, Zhongtao; Cheng, Bokun; Tse-Dinh, Yuk-Ching (2011) Crystal structure of a covalent intermediate in DNA cleavage and rejoining by Escherichia coli DNA topoisomerase I. Proc Natl Acad Sci U S A 108:6939-44
Narula, Gagandeep; Becker, Jennifer; Cheng, Bokun et al. (2010) The DNA relaxation activity and covalent complex accumulation of Mycobacterium tuberculosis topoisomerase I can be assayed in Escherichia coli: application for identification of potential FRET-dye labeling sites. BMC Biochem 11:41
Annamalai, Thirunavukkarasu; Dani, Neil; Cheng, Bokun et al. (2009) Analysis of DNA relaxation and cleavage activities of recombinant Mycobacterium tuberculosis DNA topoisomerase I from a new expression and purification protocol. BMC Biochem 10:18
Tse-Dinh, Yuk-Ching (2009) Bacterial topoisomerase I as a target for discovery of antibacterial compounds. Nucleic Acids Res 37:731-7
Cheng, Bokun; Annamalai, Thirunavukkarasu; Sorokin, Elena et al. (2009) Asp-to-Asn substitution at the first position of the DxD TOPRIM motif of recombinant bacterial topoisomerase I is extremely lethal to E. coli. J Mol Biol 385:558-67
Sorokin, Elena P; Cheng, Bokun; Rathi, Siddarth et al. (2008) Inhibition of Mg2+ binding and DNA religation by bacterial topoisomerase I via introduction of an additional positive charge into the active site region. Nucleic Acids Res 36:4788-96
Cheng, Bokun; Sorokin, Elena P; Tse-Dinh, Yuk-Ching (2008) Mutation adjacent to the active site tyrosine can enhance DNA cleavage and cell killing by the TOPRIM Gly to Ser mutant of bacterial topoisomerase I. Nucleic Acids Res 36:1017-25

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