Several inhibitors of DNA topoisomerases are clinically important anti- tumor agents. These include experimental agents such as derivatives of camptothecin, which inhibit topoisomerase I and adriamycin, etoposide and mAMSA which are topoisomerase II inhibitors. A system has been developed using the yeast Saccharomyces cerevisiae to study the mechanisms of cell killing by these agents. This system depends on mutants that enhance the permeability of yeast strains for these agents. Three major lines of experimentation will be pursued to elucidate the mechanisms of drug action. To understand the biochemical mechanisms of the interactions of the drug-with DNA topoisomerases, the yeast system will be used to screen for mutations that result in a drug resistant topoisomerase II enzyme. Mutations will be induced using in vitro mutagenesis of the yeast TOP2 gene. This will allows identification of rare mutations that lead to drug resistance. The genetic systems available in yeast allow straightforward isolation and characterization of the mutant genes. This information will be useful in understanding drug protein interactions and will be eventually useful in the design of novel anti-topoisomerase agents. Agents such as mAMSA and camptothecin are potent inducers of genetic recombination and mutations in yeast. The exact molecular nature of the recombination and mutation events will be characterized. Understanding how the anti-topoisomerase drugs induce mutations and recombination will be useful in understanding how the agents kill cells. In addition, the biochemical effects of the drugs in vivo will be characterized. The drugs stabilize an intermediate in the topoisomerase reaction, called the cleavable complex. This consists of the protein molecule covalently attached to DNA at the site of a DNA strand break introduced by the enzyme. The number of breaks introduced by various drugs will be measured, to compare how the number of breaks relates to the drug sensitivity. Studies in yeast have shown that the target of these agents are specifically DNA topoisomerases. It is not know how inhibition of topoisomerases leads to cell death. Like many other anti-cancer agents cells treated with these drugs frequently become resistant to them. In mammalian, cells, a unique type of drug resistance specific to anti- topoisomerase drugs may develop. To analyze the mechanisms of killing and resistance, mutations will be isolated that confer hypersensitivity or resistance to anti-topoisomerase drugs. The mutations will analyzed with respect to DNA repair phenotypes and effects on drug permeability. Mutations that appear to confer defects that are specific for the action of topoisomerase active drugs will be characterized, and the wild type gene will be isolated. These studies will be useful in defining pathways of cell killing and drug resistance in eukaryotic cells by these agents.
| Tombline, Gregory; Millen, Jonathan I; Polevoda, Bogdan et al. (2017) Effects of an unusual poison identify a lifespan role for Topoisomerase 2 in Saccharomyces cerevisiae. Aging (Albany NY) 9:68-97 |
| Heo, Jinho; Li, Jing; Summerlin, Matthew et al. (2015) TDP1 promotes assembly of non-homologous end joining protein complexes on DNA. DNA Repair (Amst) 30:28-37 |
| 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 |
| Nitiss, Karin C; Nitiss, John L (2014) Twisting and ironing: doxorubicin cardiotoxicity by mitochondrial DNA damage. Clin Cancer Res 20:4737-9 |
| Gao, Rui; Schellenberg, Matthew J; Huang, Shar-Yin N et al. (2014) Proteolytic degradation of topoisomerase II (Top2) enables the processing of Top2·DNA and Top2·RNA 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 |
| Hasinoff, Brian B; Wu, Xing; Nitiss, John L et al. (2012) The anticancer multi-kinase inhibitor dovitinib also targets topoisomerase I and topoisomerase II. Biochem Pharmacol 84:1617-26 |
| Nitiss, John L; Soans, Eroica; Rogojina, Anna et al. (2012) Topoisomerase assays. Curr Protoc Pharmacol Chapter 3:Unit 3.3. |
| 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 |
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