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.

Wang, Z., Ouyang, H., Tesauro, C., Ottaviani, A., He, Y., Fiorani, P., et al. (2018). Real-time analysis of cleavage and religation activity of human topoisomerase 1 based on ternary fluorescence resonance energy transfer DNA substrate. ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS, 643, 1-6 [10.1016/j.abb.2018.02.006].

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

Wang Z.;Ottaviani A.;Desideri A.
;
2018-02-16

Abstract

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.
16-feb-2018
Pubblicato
Rilevanza internazionale
Articolo
Esperti anonimi
Settore BIO/11 - BIOLOGIA MOLECOLARE
English
Enzyme activity; Fluorescence resonance energy transfer; Real-time measurement; Topoisomerase 1; Base Sequence; DNA; DNA Topoisomerases, Type I; Enzyme Assays; Fluorescent Dyes; Humans; Protein Binding; Fluorescence Resonance Energy Transfer
http://www.elsevier.com/inca/publications/store/6/2/2/7/8/7/index.htt
Wang, Z., Ouyang, H., Tesauro, C., Ottaviani, A., He, Y., Fiorani, P., et al. (2018). Real-time analysis of cleavage and religation activity of human topoisomerase 1 based on ternary fluorescence resonance energy transfer DNA substrate. ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS, 643, 1-6 [10.1016/j.abb.2018.02.006].
Wang, Z; Ouyang, H; Tesauro, C; Ottaviani, A; He, Y; Fiorani, P; Xie, H; Desideri, A; Fu, Z
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/2108/228377
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