DNA topoisomerases (Topo) are essential enzymes required to maintain the superhelical topology of genomic DNA during the processes of DNA replication, transcription and chromosome segregation. All topoisomerases are united by the mechanistic feature of using an active site tyrosine to attack the DNA backbone and form a dynamic covalent phosphotyrosine linkage and flexible strand nick that is of extraordinary importance in DNA metabolism and pharmacology. Topoisomerases have long been targets for drugs that bind to and stabilize the covalent complex, but mechanistically novel classes of compounds have not been discovered in more than two decades. This competitive renewal seeks to elucidate how the dynamics and mobility of the enzyme DNA covalent complex facilitate the various DNA transformations catalyzed by the enzyme, including drug binding. We also aim to develop a chemical platform to discover new classes of small molecule ligands that inhibit or poison human, bacterial and parasite type I Topo's. The significance of this work is linking the essential dynamic features of these enzymes to biological function and drug action.
The aims are to: (i) Understand how the dynamic mobility of the topoisomerase IB DNA complex leads to supercoil relaxation and drug inhibition. The free strand rotation model for DNA relaxation by Topo IB requires that the DNA segment 3'to the covalent attachment is transiently released from its noncovalent interactions with the enzyme to allow rotation of the DNA around the superhelical axis. Using 19F NMR relaxation methods, we will investigate the dynamics of the covalently bound DNA using novel substrates that are specifically labeled with 19F in rigid and dynamic regions of the DNA complex. (ii) Understand how the dynamic mobility of the topoisomerase IB DNA complex leads to recombinogenic DNA strand exchange reactions. The facile strand exchange reactions promoted by Topo also necessitate dynamic and thermodynamic destabilization of the DNA duplex near to the cleavage site. We will explore these key mechanistic aspects using novel NMR imino proton exchange measurements, and stopped flow kinetic and thermodynamic measurements. (iii) Develop chemical tools to rapidly profile the activities of topoisomerases and discover new ligands that modulate Topo function. A complete repertoire of high throughput topoisomerase screens will be developed to facilitate the discovery of novel small molecule topoisomerase inhibitors or poisons. These methods will provide new profiling tools for topoisomerase activity in clinical samples.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM068626-07
Application #
7847423
Study Section
Molecular Genetics A Study Section (MGA)
Program Officer
Gerratana, Barbara
Project Start
2003-06-01
Project End
2012-05-31
Budget Start
2010-06-01
Budget End
2011-05-31
Support Year
7
Fiscal Year
2010
Total Cost
$284,130
Indirect Cost
Name
Johns Hopkins University
Department
Pharmacology
Type
Schools of Medicine
DUNS #
001910777
City
Baltimore
State
MD
Country
United States
Zip Code
21218
Mak, Lok Hang; Knott, Jessica; Scott, Katherine A et al. (2012) Arylstibonic acids are potent and isoform-selective inhibitors of Cdc25a and Cdc25b phosphatases. Bioorg Med Chem 20:4371-6
Jun, Helen; Stivers, James T (2012) Diverse energetic effects of charge reversal mutations of poxvirus topoisomerase IB. Biochemistry 51:2940-9
Lauzon, Carolyn B; van Zijl, Peter; Stivers, James T (2011) Using the water signal to detect invisible exchanging protons in the catalytic triad of a serine protease. J Biomol NMR 50:299-314
Ye, Yu; Stivers, James T (2010) Fluorescence-based high-throughput assay for human DNA (cytosine-5)-methyltransferase 1. Anal Biochem 401:168-72
Stahley, Mary R; Stivers, James T (2010) Mechanism and specificity of DNA strand exchange catalyzed by vaccinia DNA topoisomerase type I. Biochemistry 49:2786-95
Kim, Hyeongnam; Cardellina 2nd, John H; Akee, Rhone et al. (2008) Arylstibonic acids: novel inhibitors and activators of human topoisomerase IB. Bioorg Chem 36:190-7
Stivers, James T (2008) Small molecule versus DNA repair nanomachine. Nat Chem Biol 4:86-8
Nagarajan, Rajesh; Stivers, James T (2007) Unmasking Anticooperative DNA-binding interactions of vaccinia DNA topoisomerase I. Biochemistry 46:192-9
Bond, Alexis; Reichert, Zachary; Stivers, James T (2006) Novel and specific inhibitors of a poxvirus type I topoisomerase. Mol Pharmacol 69:547-57
Stivers, James T; Nagarajan, Rajesh (2006) Probing enzyme phosphoester interactions by combining mutagenesis and chemical modification of phosphate ester oxygens. Chem Rev 106:3443-67

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