New technique for uncoupling the cleavage and religation reactions of eukaryotic topoisomerase I. The mode of action of camptothecin at a specific recognition site.
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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DOI：
10.1016/0022-2836(91)90503-x
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发表时间：
1991-12
影响因子：
5.6
通讯作者：
Jesper Q. Svejstrup;K. Christiansen;Irina Gromova;A. H. Andersen;O. Westergaard
中科院分区：
文献类型：
--
作者：
Jesper Q. Svejstrup;K. Christiansen;Irina 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 3m-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 antineoplastic 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.
