title: 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.
abstract: Human DNA topoisomerase I (hTopI) is a nuclear enzyme that catalyzes relaxation of super helical tension that arises in the genome during essential DNA metabolic processes. This is accomplished through a common reaction mechanism shared among the type IB topoisomerase enzymes, including eukaryotic and poxvirus topoisomerase I. The mechanism of hTopI is specifically targeted in cancer treatment using camptothecin derivatives. These drugs convert the hTopI activity into a cellular poison, and hence the cytotoxic effects of camptothecin derivatives correlate with the hTopI activity. Therefore, fast and reliable techniques for high throughput measurements of hTopI activity are of high clinical interest. Here we demonstrate potential applications of a fluorophore-quencher based DNA sensor designed for measurement of hTopI cleavage-ligation activities, which are the catalytic steps affected by camptothecin. The kinetic analysis of the hTopI reaction with the DNA sensor exhibits a characteristic burst profile. This is the result of a two-step ping-pong reaction mechanism, where a fast first reaction, the one creating the signal, is followed by a slower second reaction necessary for completion of the catalytic cycle. Hence, the burst profile holds information about two reactions in the enzymatic mechanism. Moreover, it allows the amount of active enzyme in the reaction to be determined. The presented results pave the way for future high throughput drug screening and the potential of measuring active hTopI concentrations in clinical samples for individualized treatment.
authors.affiliation: Department of Molecular Biology and Genetics, Aarhus University, Denmark. brk@mbg.au.dk.
authors.forename: Emil L
authors.initials: EL
authors.lastname: Kristoffersen
authors.forename: Line A
authors.initials: LA
authors.lastname: Jørgensen
authors.forename: Oskar
authors.initials: O
authors.lastname: Franch
authors.forename: Michael
authors.initials: M
authors.lastname: Etzerodt
authors.forename: Rikke
authors.initials: R
authors.lastname: Frøhlich
authors.forename: Lotte
authors.initials: L
authors.lastname: Bjergbæk
authors.forename: Magnus
authors.initials: M
authors.lastname: Stougaard
authors.forename: Yi-Ping
authors.initials: YP
authors.lastname: Ho
authors.forename: Birgitta R
authors.initials: BR
authors.lastname: Knudsen
mesh_terms: D055162:Biocatalysis; D004247:DNA / Q000378:metabolism*; D004264:DNA Topoisomerases, Type I / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D060888:Real-Time Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis; D013379:Substrate Specificity
chemical_list: D005456:Fluorescent Dyes; D011994:Recombinant Proteins; D004247:DNA; D004264:DNA Topoisomerases, Type I; C528909:TOP1 protein, human