Topoisomerase II (Top2) is the target of a wide range of active anti-cancer agents. Drugs targeting Top2 are widely used and active agents, but a detailed understanding of how they act against the enzyme has proven elusive. Clinically active Top2 targeting agents lead to enzyme mediated DNA damage, and are referred to as Top2 poisons. Detailed information about the action of Top2 poisons has the potential for leading to safer and more active anti-cancer drugs. An important model system for understanding the biological and biochemical properties of DNA topoisomerases is the yeast Saccharomyces cerevisiae. The genetic tools available in this organism also make it a powerful system for studying mechanisms of action of anti-cancer drugs targeting topoisomerases. Yeast Top2 has also been the enzyme of choice for structural studies of eukaryotic Top2. The overall goal of this project is to apply all of the tools available with the yeast system to obtain a detailed understanding of how Top2 poisons interact with the enzyme, and how Top2 drug interactions lead to Top2 mediated DNA damage. We propose a combination of genetic, biochemical and structural approaches to understand the action of Top2 poisons on their target enzymes. Genetic studies will allow the isolation and characterization of novel drug hypersensitive mutants of Top2. These studies will increase our understanding of how Top2 poisons bind to the enzyme and generate enzyme-mediated DNA damage. Assays for assessing drug action, including measurements of drug affinity and enzyme mediated DNA cleavage will be applied to test how drug hypersensitivity occurs in the mutant proteins. We also plan to study a novel mutant of human Top2 that mimics the action of Top2 poisons in the absence of drug. Studies with this mutant will provide a biochemical understanding of Top2 mediated DNA damage in the context of the overall reaction cycle. Finally, the drug hypersensitive mutants will provide necessary reagents for structural studies that will attempt to solve the structure of drug:protein:DNA ternary complexes. The proposed experiments will be useful in the design of new agent targeting Top2, and may suggest more effective use of Top2 targeting drugs that are in clinical use.

Public Health Relevance

Topoisomerase II (Top2) is the target of active anti-cancer agents that in commonly use. While these drugs have been in use for many years, a detailed understanding of how they act against the Top2 has proven elusive. The proposed studies will provide a detailed picture of how Top2 targeting drugs interact with the enzyme, and may lead to tools needed for designing safer and more effective drugs that target this enzyme.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA052814-19
Application #
8213486
Study Section
Basic Mechanisms of Cancer Therapeutics Study Section (BMCT)
Program Officer
Arya, Suresh
Project Start
1997-06-01
Project End
2015-01-31
Budget Start
2012-02-01
Budget End
2013-01-31
Support Year
19
Fiscal Year
2012
Total Cost
$282,872
Indirect Cost
$91,007
Name
University of Illinois at Chicago
Department
Pharmacology
Type
Schools of Pharmacy
DUNS #
098987217
City
Chicago
State
IL
Country
United States
Zip Code
60612
Nitiss, Karin C; Nitiss, John L (2014) Twisting and ironing: doxorubicin cardiotoxicity by mitochondrial DNA damage. Clin Cancer Res 20:4737-9
Katyal, Sachin; Lee, Youngsoo; Nitiss, Karin C et al. (2014) Aberrant topoisomerase-1 DNA lesions are pathogenic in neurodegenerative genome instability syndromes. Nat Neurosci 17:813-21
Gao, Rui; Schellenberg, Matthew J; Huang, Shar-Yin N et al. (2014) Proteolytic degradation of topoisomerase II (Top2) enables the processing of Top2┬ĚDNA and Top2┬ĚRNA covalent complexes by tyrosyl-DNA-phosphodiesterase 2 (TDP2). J Biol Chem 289:17960-9
Nitiss, John L; Nitiss, Karin C (2013) Tdp2: a means to fixing the ends. PLoS Genet 9:e1003370
Bahmed, Karim; Nitiss, Karin C; Nitiss, John L (2010) Yeast Tdp1 regulates the fidelity of nonhomologous end joining. Proc Natl Acad Sci U S A 107:4057-62
Nitiss, John L (2009) Targeting DNA topoisomerase II in cancer chemotherapy. Nat Rev Cancer 9:338-50
Rogojina, Anna T; Nitiss, John L (2008) Isolation and characterization of mAMSA-hypersensitive mutants. Cytotoxicity of Top2 covalent complexes containing DNA single strand breaks. J Biol Chem 283:29239-50
He, Xiaoping; van Waardenburg, Robert C A M; Babaoglu, Kerim et al. (2007) Mutation of a conserved active site residue converts tyrosyl-DNA phosphodiesterase I into a DNA topoisomerase I-dependent poison. J Mol Biol 372:1070-81
Nitiss, Karin C; Malik, Mobeen; He, Xiaoping et al. (2006) Tyrosyl-DNA phosphodiesterase (Tdp1) participates in the repair of Top2-mediated DNA damage. Proc Natl Acad Sci U S A 103:8953-8
Hasinoff, Brian B; Wu, Xing; Krokhin, Oleg V et al. (2005) Biochemical and proteomics approaches to characterize topoisomerase IIalpha cysteines and DNA as targets responsible for cisplatin-induced inhibition of topoisomerase IIalpha. Mol Pharmacol 67:937-47

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