Svejstrup J Q, Christiansen K, Gromova I I, Andersen A H, Westergaard O
Department of Molecular Biology and Plant Physiology, University of Aarhus, Denmark.
J Mol Biol. 1991 Dec 5;222(3):669-78. doi: 10.1016/0022-2836(91)90503-x.
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.
一种用于在特定位点解开拓扑异构酶I的切割和重新连接半反应的新技术已被开发出来。该技术利用一种自杀性DNA底物来实现酶介导的切割而不伴随重新连接。随后，通过添加一种能够与自杀性DNA底物的未切割链杂交的寡核苷酸，可以实现有效的重新连接。该技术被用于研究不同化合物对拓扑异构酶I半反应的影响。结果表明，高于200 mM的NaCl浓度会抑制拓扑异构酶I介导的切割，而高达3 M-NaCl的浓度似乎对重新连接反应没有影响。发现二价阳离子Mg2+、Ca2+和Mn2+会增强拓扑异构酶I的切割但不增强其重新连接反应，而Cu2+和Zn2+则抑制这两种反应。此外，还研究了抗肿瘤药物喜树碱对拓扑异构酶I半反应的影响。发现该药物在所研究的位点不影响拓扑异构酶I的切割反应，而酶的重新连接反应受到抑制。还发现，即使在复合物形成后添加该药物，喜树碱也能稳定酶-DNA切割复合物。
