The long-term objective of this project is to understand how type IB DNA topoisomerases (topo) catalyze reversible DNA strand cleavage and religation, and how the chemical and dynamic nature of the phosphotyrosyI-DNA covalent complex promotes such important biological processes as DNA strand transfer, recombination, supercoil unwinding, and anticancer drug binding. In this work, we will employ the small, sequence specific type 1B topo from vaccinia virus because it is the only type IB enzyme amenable to detailed NMR, fluorescence, and kinetic and thermodynamic studies.
The specific aims are as follows: (i) Determine the basis for specific recognition of CCCTT sites in DNA. The importance of base and phosphodiester interactions in specificity will be evaluated using novel base analogs and nonbridging methylphosphonate substitutions, respectively. (ii) Elucidate the mechanism of nucleophilic catalysis. The catalytic interactions of the scissile phosphodiester will be dissected using the combined approach of mutagenesis, nonbridging phosphorothioate substitutions. Novel solid-state REDOR NMR structural methods will be used to confirm these interactions. (iii) Understand how Topo I removes supercoils from DNA. Topo I must release its grip on DNA to allow supercoil relaxation to occur. To elucidate these critical motions, 19F-labeled DNA molecules and NMR spectroscopy will be used to measure the dynamics of the DNA within the covalent complex. In addition, we will use a small supercoiled plasmid with a single cleavage site, to evaluate the role of three key variables on the supercoil relaxation mechanism: the DNA superhelical density, the lifetime of the covalent complex, and the """"""""tightness"""""""" of the enzyme grip on the rotating DNA. We anticipate these measurements will guide our efforts to design inhibitors of Topo I and to engineer the enzyme to perform other useful DNA transformations.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM068626-04
Application #
7059445
Study Section
Physical Biochemistry Study Section (PB)
Program Officer
Ikeda, Richard A
Project Start
2003-06-01
Project End
2008-05-31
Budget Start
2006-06-01
Budget End
2008-05-31
Support Year
4
Fiscal Year
2006
Total Cost
$271,418
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
Feng, You; Xie, Nan; Jin, Miyeong et al. (2011) A transient kinetic analysis of PRMT1 catalysis. Biochemistry 50:7033-44
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
Stivers, James T (2008) Small molecule versus DNA repair nanomachine. Nat Chem Biol 4:86-8
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
Nagarajan, Rajesh; Stivers, James T (2007) Unmasking Anticooperative DNA-binding interactions of vaccinia DNA topoisomerase I. Biochemistry 46:192-9
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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