Real-time investigation of human topoisomerase I reaction kinetics using an optical sensor: a fast method for drug screening and determination of active enzyme concentrations

Kristoffersen, Emil L.; Jørgensen, Line A.; Franch, Oskar; Etzerodt, Michael; Frøhlich, Rikke; Bjergbæk, Lotte; Stougaard, Magnus; Ho, Yi‐Ping; Knudsen, Birgitta R.

doi:10.1039/c5nr01474c

Cited by 14 publications

(6 citation statements)

References 33 publications

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“…However, several type IB topoisomerases, which differ from type IA enzymes in forming a cleavage intermediate in which the cleaved strand is covalently linked to the active site of the enzyme, have been subjected to kinetic analyses. The rate constants of template cleavage and religation for human topoisomerase I at 37 °C are 69 × 10 −4 and 9.5 × 10 −4 s −1 , respectively 20 , whereas the two parameters for Vaccinia topoisomerase I at 37 °C are 0.04 and 0.12 s −1 , respectively 21 . By comparison, the k clv and the k reli of Sso topo III at 37 °C were 0.0291 and 1.49 × 10 −3 s −1 , respectively.…”

Section: Discussionmentioning

confidence: 99%

Kinetic insights into the temperature dependence of DNA strand cleavage and religation by topoisomerase III from the hyperthermophile Sulfolobus solfataricus

Zhang

Pan

et al. 2017

Sci Rep

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All cellular organisms encode type IA topoisomerases which catalyze DNA topological changes essential for DNA transactions. However, the kinetics of the reaction catalyzed by these enzymes remains poorly characterized. Here we measured the rapid kinetics of template binding, cleavage and religation by Sso topo III, a type IA topoisomerase from the hyperthermophilic archaeon Sulfolobus solfataricus, by using a novel FRET/PIFE-based method in a stopped-flow spectrometer. We show that Sso topo III bound the template rapidly, and the rate of binding was 2-3 orders of magnitudes higher than that of template cleavage at 25 °C. The rate of template cleavage was favored over that of template religation by the enzyme, and was more so at lower temperatures (25-55 °C). Significant template cleavage [(2.23 ± 0.11) × 10 −3 s −1 ] was observed while little religation was detectable at 25 °C. This is consistent with the presence of a higher activation energy for template religation (41 ± 5 kcal·mol −1 ) than that for template cleavage (32 ± 1 kcal·mol −1 ). Our results provide a kinetic interpretation for the ability of Sso topo III to relax negatively supercoiled DNA only at higher temperature and offer clues to the adaptation of the reaction mechanisms of thermophilic enzymes to high temperature.DNA topoisomerases are ubiquitous enzymes that catalyze topological changes in DNA essential for DNA transactions, such as DNA replication, transcription, DNA repair and recombination 1 . Type IA topoisomerases exist in all cellular organisms and include topoisomerases I and III as well as reverse gyrase 1 . These enzymes are capable of relaxing negative DNA supercoils, or introducing positive DNA supercoils in the case of reverse gyrase. Much of our current knowledge about the mechanistic aspects of type IA topoisomerases is derived from the studies of Escherichia coli topoisomerase I, a prototype of this subclass of topoisomerases 2, 3 . According to a well-accepted model, type IA topoisomerases relax DNA using an enzyme-bridged mechanism, which entails four steps in a catalytic cycle 4,5 . The topoisomerase binds non-covalently to one strand in the single-stranded regions of DNA (step 1), and cleaves the strand non-randomly with the concomitant formation of a covalent linkage between the active-site tyrosyl of the enzyme and the scissile phosphate (step 2); the complementary strand passes through the transient opening on the cleaved strand (step 3), followed by the religation of the cleaved strand (step 4).Topoisomerase III from the hyperthermophilic archaeon Sulfolobus solfataricus (Sso topo III) is a type IA topoisomerase. Sso topo III is most active in DNA relaxation at 75 °C, a temperature optimal for the growth of the

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Section: Discussionmentioning

confidence: 99%

Kinetic insights into the temperature dependence of DNA strand cleavage and religation by topoisomerase III from the hyperthermophile Sulfolobus solfataricus

Zhang

Pan

et al. 2017

Sci Rep

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“…Human topoisomerase I was expressed and purified essentially as described (8). Expression and purification of human Topoisomerase IIα (Topo IIα) was as described in (57).…”

Section: Methodsmentioning

confidence: 99%

Interlinked DNA nano-circles for measuring topoisomerase II activity at the level of single decatenation events

Kristoffersen

Givskov

Jørgensen

et al. 2017

Nucleic Acids Research

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DNA nano-structures present appealing new means for monitoring different molecules. Here, we demonstrate the assembly and utilization of a surface-attached double-stranded DNA catenane composed of two intact interlinked DNA nano-circles for specific and sensitive measurements of the life essential topoisomerase II (Topo II) enzyme activity. Topo II activity was detected via the numeric release of DNA nano-circles, which were visualized at the single-molecule level in a fluorescence microscope upon isothermal amplification and fluorescence labeling. The transition of each enzymatic reaction to a micrometer sized labeled product enabled quantitative detection of Topo II activity at the single decatenation event level rendering activity measurements in extracts from as few as five cells possible. Topo II activity is a suggested predictive marker in cancer therapy and, consequently, the described highly sensitive monitoring of Topo II activity may add considerably to the toolbox of individualized medicine where decisions are based on very sparse samples.

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“…Although various sensors have been published for measurement of TOP1 [ 49 , 50 ] and TDP1 [ 51 , 52 , 53 , 54 ], most in vitro TOP1 and TDP1 drug-screening approaches still use gel-based assays for measuring TDP1 and TOP1 activity [ 55 , 56 , 57 ]. Although the usability of gel-based assays is well documented, they are time-consuming and not well suited for high-throughput screening.…”

Section: Introductionmentioning

confidence: 99%

A Dual-Sensor-Based Screening System for In Vitro Selection of TDP1 Inhibitors

Jakobsen

Keller

González

et al. 2021

Sensors

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DNA sensors can be used as robust tools for high-throughput drug screening of small molecules with the potential to inhibit specific enzymes. As enzymes work in complex biological pathways, it is important to screen for both desired and undesired inhibitory effects. We here report a screening system utilizing specific sensors for tyrosyl-DNA phosphodiesterase 1 (TDP1) and topoisomerase 1 (TOP1) activity to screen in vitro for drugs inhibiting TDP1 without affecting TOP1. As the main function of TDP1 is repair of TOP1 cleavage-induced DNA damage, inhibition of TOP1 cleavage could thus reduce the biological effect of the TDP1 drugs. We identified three new drug candidates of the 1,5-naphthyridine and 1,2,3,4-tetrahydroquinolinylphosphine sulfide families. All three TDP1 inhibitors had no effect on TOP1 activity and acted synergistically with the TOP1 poison SN-38 to increase the amount of TOP1 cleavage-induced DNA damage. Further, they promoted cell death even with low dose SN-38, thereby establishing two new classes of TDP1 inhibitors with clinical potential. Thus, we here report a dual-sensor screening approach for in vitro selection of TDP1 drugs and three new TDP1 drug candidates that act synergistically with TOP1 poisons.

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Real-time investigation of human topoisomerase I reaction kinetics using an optical sensor: a fast method for drug screening and determination of active enzyme concentrations

Cited by 14 publications

References 33 publications

Kinetic insights into the temperature dependence of DNA strand cleavage and religation by topoisomerase III from the hyperthermophile Sulfolobus solfataricus

Kinetic insights into the temperature dependence of DNA strand cleavage and religation by topoisomerase III from the hyperthermophile Sulfolobus solfataricus

Interlinked DNA nano-circles for measuring topoisomerase II activity at the level of single decatenation events

A Dual-Sensor-Based Screening System for In Vitro Selection of TDP1 Inhibitors

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