Abstract
Tyrosyl-DNA phosphodiesterase I (TDP1) hydrolyzes the drug-stabilized 3’phospho-tyrosyl bond formed between DNA topoisomerase I (TOPO1) and DNA. TDP1-mediated hydrolysis uses a nucleophilic histidine (Hisnuc) and a general acid/base histidine (Hisgab). A Tdp1Hisgab to Arg mutant identified in patients with the autosomal recessive neurodegenerative disease SCAN1 causes stabilization of the TDP1-DNA intermediate. Based on our previously reported Hisgab-substitutions inducing yeast toxicity (Gajewski et al. J. Mol. Biol. 415, 741-758, 2012), we propose that converting TDP1 into a cellular poison by stabilizing the covalent enzyme-DNA intermediate is a novel therapeutic strategy for cancer treatment. Here, we analyzed the toxic effects of two TDP1 catalytic mutants in HEK293 cells. Expression of human Tdp1HisnucAla and Tdp1HisgabAsn mutants results in stabilization of the covalent TDP1-DNA intermediate and induces cytotoxicity. Moreover, these mutants display reduced in vitro catalytic activity compared to wild type. Co-treatment of Tdp1mutant with topotecan shows more than additive cytotoxicity. Overall, these results support the hypothesis that stabilization of the TDP1-DNA covalent intermediate is a potential anti-cancer therapeutic strategy.
Highlights
Tyrosyl-DNA phosphodiesterase I (TDP1) hydrolyzes 3’phospho-adducts and to a limited extent 5’phospho-adducts within DNA strand breaks
The expression level of the catalytic mutants was similar to wild type, suggesting that the phenotypes are due to alterations in catalytic function rather than a difference in expression (Figure 2B) or expression levels of yeast and human TOPO1
Our reported yeast observations suggested that TDP1 is a suitable target to turn a DNA repair enzyme into a cellular toxin as a potential novel anti-cancer treatment strategy [3, 4, 32]
Summary
Tyrosyl-DNA phosphodiesterase I (TDP1) hydrolyzes 3’phospho-adducts and to a limited extent 5’phospho-adducts within DNA strand breaks (reviewed in [1]). Tdp catalysis is structurally and mechanistically conserved from yeast (y) to human (h), and centers on the formation and resolution of a requisite covalent TDP1-DNA reaction intermediate, called a TDP1DNA covalent complex (TDP1-cc) (Figure 1) This two-step cycle utilizes spatial and temporal coordinated action of two catalytic histidines [2,3,4,5,6,7,8]. Besides the DNA repair enzyme TDP1, DNA topoisomerases (TOPOs; TOPO is used instead of the official abbreviation “TOP” to emphasize the difference between the protein names TDP1 and TOP1) that adjust DNA topology form a transient covalent enzyme-DNA reaction intermediate (TOPO-cc) during their catalytic cycle (review in [9,10,11]) This transient covalent enzymeDNA intermediate can potentially result in toxic DNA lesions. This paradigm is extensively exploited by cancerchemotherapeutics that induce toxicity via stabilization of the enzyme-DNA intermediate, e.g. etoposide for TOPO2 and camptothecin (CPT) for TOPO1, including FDAapproved CPT analogs topotecan and irinotecan [12, 13]
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