Tyrosyl-DNA Phosphodiesterase I Catalytic Mutants Reveal an Alternative Nucleophile That Can Catalyze Substrate Cleavage

Comeaux, Evan Q.; Cuya, Selma M.; Kojima, Kensuke; Jafari, Nauzanene; Wanzeck, Keith; Mobley, James A.; Bjornsti, Mary-Ann; Waardenburg, Robert C.A.M. van

doi:10.1074/jbc.m114.635284

Cited by 18 publications

(43 citation statements)

References 45 publications

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“…However, there was no reduction in CIN when the catalytically inactive Tdp1 H182A mutant was overexpressed as we expected. This observation is supported by recent findings where Tdp1 H182A retains catalytic activity in vivo (Comeaux et al, 2015). This experiment suggests that it is not the increased enzymatic activity of Tdp1 that is responsible for the dCIN phenotypes.…”

Section: Rhabdomyosarcoma Lines Expressing Higher Levels Of Tdp1 Showsupporting

confidence: 85%

Systematic over-expression screens for chromosome instability identify conserved dosage chromosome instability genes in yeast and human tumors

Duffy

Fam

Wang

et al. 2016

Preprint

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Somatic copy number amplifications (SCNAs) and gene over-expression are common features of many cancers. To determine the role of gene over-expression on genome stability, we performed functional genomic screens in the budding yeast for chromosome instability, a defining characteristic of cancer that can be targeted by therapeutics. Over-expression of 245 yeast genes increases chromosome instability by influencing processes such as chromosome segregation and DNA damage repair. Testing candidate human homologs, which were highly recurrently altered in tumors lead to the identification of 2 genes, Tdp1 and Taf12 that contribute to CIN in human cells when over-expressed.Rhabdomyosarcoma lines with higher levels of Tdp1 also show chromosome instability and can be partially rescued by siRNA-mediated knockdown of Tdp1. Using synthetic dosage lethality screens in yeast, we identified candidate target genes that will specifically target tumors with high levels of Tdp1.We demonstrate the utility of functional genetic screens in model organisms to broaden the spectrum of CIN genes, to identify novel genes relevant to chromosome instability in humans and to identify candidate gene targets that can be leveraged to selectively kill tumors over-expressing specific genes.

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Section: Rhabdomyosarcoma Lines Expressing Higher Levels Of Tdp1 Showsupporting

confidence: 85%

Systematic over-expression screens for chromosome instability identify conserved dosage chromosome instability genes in yeast and human tumors

Duffy

Fam

Wang

et al. 2016

Preprint

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“…First, we detected catalytic activity in vitro and a cellular phenotype with the reported catalytic inactive hTdp1H 263 A mutant [5, 8]. We recently reported a similar observation for the analogous yeast yTdp1H 182 A mutant [32]. We concluded that the His preceding the His nuc is able to rotate into the catalytic pocket in case His nuc is substituted for Ala [3].…”

Section: Discussionmentioning

confidence: 83%

Dysregulated human Tyrosyl-DNA phosphodiesterase I acts as cellular toxin

et al. 2016

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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.

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“…88 In hTdp1, this neighboring residue is H262, which is not included in the currently reported QM cluster model. The exploration of Tdp1 in the context of SCAN1 therapy and active site mutations with larger models (QM and layered QM/ MM models) are planned for future studies.…”

Section: ■ Conclusionmentioning

confidence: 94%

A Theoretical Study of Phosphoryl Transfers of Tyrosyl-DNA Phosphodiesterase I (Tdp1) and the Possibility of a “Dead-End” Phosphohistidine Intermediate

DeYonker

Webster

2015

Biochemistry

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Tyrosyl-DNA phosphodiesterase I (Tdp1) is a DNA repair enzyme conserved across eukaryotes that catalyzes the hydrolysis of the phosphodiester bond between the tyrosine residue of topoisomerase I and the 3'-phosphate of DNA. Atomic level details of the mechanism of Tdp1 are proposed and analyzed using a fully quantum mechanical, geometrically constrained model. The structural basis for the computational model is the vanadate-inhibited crystal structure of human Tdp1 (hTdp1, Protein Data Bank entry 1RFF ). Density functional theory computations are used to acquire thermodynamic and kinetic data along the catalytic pathway, including the phosphoryl transfer and subsequent hydrolysis. Located transition states and intermediates along the reaction coordinate suggest an associative phosphoryl transfer mechanism with five-coordinate phosphorane intermediates. Similar to both theoretical and experimental results for phospholipase D, the proposed mechanism for hTdp1 also includes the thermodynamically favorable possibility of a four-coordinate phosphohistidine "dead-end" product.

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Tyrosyl-DNA Phosphodiesterase I Catalytic Mutants Reveal an Alternative Nucleophile That Can Catalyze Substrate Cleavage

Cited by 18 publications

References 45 publications

Systematic over-expression screens for chromosome instability identify conserved dosage chromosome instability genes in yeast and human tumors

Systematic over-expression screens for chromosome instability identify conserved dosage chromosome instability genes in yeast and human tumors

Dysregulated human Tyrosyl-DNA phosphodiesterase I acts as cellular toxin

A Theoretical Study of Phosphoryl Transfers of Tyrosyl-DNA Phosphodiesterase I (Tdp1) and the Possibility of a “Dead-End” Phosphohistidine Intermediate

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