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Real-time analysis of cleavage and religation activity of human topoisomerase 1 based on ternary fluorescence resonance energy transfer DNA substrate

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  • Zhenxing Wang, Southwest University, Universita degli Studi di Roma Tor Vergata, Department of Oral and Maxillofacial Surgery, Xiangya Hospital and School of Stomatology, Central South University
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  • Hui Ouyang, Southwest University
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  • Cinzia Tesauro
  • Alessio Ottaviani, Universita degli Studi di Roma Tor Vergata
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  • Yong He, Southwest University
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  • Paola Fiorani, Consiglio Nazionale delle Ricerche
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  • Hui Xie, Department of Oral and Maxillofacial Surgery, Xiangya Hospital and School of Stomatology, Central South University
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  • Alessandro Desideri, Department of Biology, University of “Tor Vergata”, Rome
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  • Zhifeng Fu, Southwest University

Human topoisomerase 1B is a ubiquitous and essential enzyme involved in relaxing the topological state of supercoiled DNA to allow the progression of fundamental DNA metabolism. Its enzymatic catalytic cycle consists of cleavage and religation reaction. A ternary fluorescence resonance energy transfer biosensor based on a suicide DNA substrate conjugated with three fluorophores has been developed to monitor both cleavage and religation Topoisomerase I catalytic function. The presence of fluorophores does not alter the specificity of the enzyme catalysis on the DNA substrate. The enzyme-mediated reaction can be tracked in real-time by simple fluorescence measurement, avoiding the use of risky radioactive substrate labeling and time-consuming denaturing gel electrophoresis. The method is applied to monitor the perturbation brought by single mutation on the cleavage or religation reaction and to screen the effect of the camptothecin anticancer drug monitoring the energy transfer decrease during religation reaction. Pathological mutations usually affect only the cleavage or the religation reaction and the proposed approach represent a fast protocol for assessing chemotherapeutic drug efficacy and analyzing mutant's properties.

Original languageEnglish
JournalArchives of Biochemistry and Biophysics
Volume643
Pages (from-to)1-6
Number of pages6
ISSN0003-9861
DOIs
Publication statusPublished - 16 Feb 2018

    Research areas

  • Enzyme activity, Fluorescence resonance energy transfer, Real-time measurement, Topoisomerase 1

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