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New technique for uncoupling the cleavage and religation reactions of eukaryotic topoisomerase I. The mode of action of camptothecin at a specific recognition site

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  • J Q Svejstrup, Department of Molecular Biology and Plant Physiology, University of Aarhus, Denmark.
  • ,
  • K Christiansen
  • ,
  • I I Gromova
  • ,
  • A H Andersen
  • O Westergaard

A new technique for uncoupling the cleavage and religation half-reactions of topoisomerase I at a specific site has been developed. The technique takes advantage of a suicidal DNA substrate to attain enzyme-mediated cleavage without concomitant religation. Efficient religation can be achieved, subsequently, by addition of an oligonucleotide capable of hybridising to the non-cleaved strand of the suicide DNA substrate. The technique was used to study the effect of different compounds on the half-reactions of topoisomerase I. It was shown that topoisomerase I-mediated cleavage was inhibited by NaCl concentrations higher than 200 mM, while the religation reaction seemed unaffected by concentrations as high as 3 M-NaCl. The divalent cations Mg2+, Ca2+ and Mn2+ were found to enhance the cleavage but not the religation reaction of topoisomerase I, whereas Cu2+ and Zn2+ inhibited both reactions. Furthermore, the effect of the anti-neoplastic agent, camptothecin, on the half-reactions of topoisomerase I was investigated. It was found that the drug did not affect the cleavage reaction of topoisomerase I at the studied site, while the religation reaction of the enzyme was inhibited. Camptothecin was found to stabilise the enzyme-DNA cleavage complex even when the drug was added after complex formation.

Original languageEnglish
JournalJournal of Molecular Biology
Volume222
Issue3
Pages (from-to)669-78
Number of pages10
ISSN0022-2836
Publication statusPublished - 5 Dec 1991

    Research areas

  • Antineoplastic Agents, Binding Sites, Camptothecin, Cations, Divalent, DNA, DNA Topoisomerases, Type I, DNA, Single-Stranded, Eukaryotic Cells, Humans, Sodium Chloride, Journal Article, Research Support, Non-U.S. Gov't

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