Coderoni S, Paparelli M, Gianfranceschi G L
Department of Molecular, Cellular and Animal Biology, University of Camerino, Italy.
Mol Biol Rep. 1993 Feb;17(2):129-34. doi: 10.1007/BF00996220.
The effects of CPT on the calf thymus Topoisomerase I-mediated DNA breakage-reunion reaction were studied at an enzyme concentration range proper for evidencing, at the same time, both DNA relaxation and DNA cleavage/religation. Some of the requirements and the optimal conditions for the formation and reversal of the CPT-trapped Topoisomerase I-DNA cleavable complex are also characterized. We conclude that: 1. Calf thymus (100 kDa) Topoisomerase I requires, for maximal DNA cleavage activity, specific and characteristic reaction conditions. 2. CPT does not affect these optimal conditions, but only stabilizes the normal enzyme-DNA intermediate. In this way, the drug lowers the religation process, becoming responsible for the relaxation inhibition. 3. The optimum of monovalent salt concentration for cleavable complex formation is found between 30 and 70 mM. These values are lower than those required for the relaxation activity optimum (75-125 mM NaCl). 4. The addition of 0.5 M monovalent salt causes reversal of the reaction, and shifts the equilibrium distribution between cleavable intermediate and closed relaxed DNA in the direction of DNA resealing. Therefore, it is suggested that salt affects the cleavage but not the religation reaction.
在适合同时证明DNA松弛和DNA切割/连接的酶浓度范围内，研究了喜树碱（CPT）对小牛胸腺拓扑异构酶I介导的DNA断裂-重连反应的影响。还对喜树碱捕获的拓扑异构酶I-DNA可切割复合物形成和逆转的一些要求及最佳条件进行了表征。我们得出以下结论：1. 小牛胸腺（100 kDa）拓扑异构酶I要实现最大DNA切割活性，需要特定且具有特征性的反应条件。2. 喜树碱不影响这些最佳条件，只是稳定了正常的酶-DNA中间体。通过这种方式，该药物降低了重连过程，导致松弛抑制。3. 可切割复合物形成的单价盐最佳浓度在30至70 mM之间。这些值低于松弛活性最佳状态所需的值（75 - 125 mM NaCl）。4. 添加0.5 M单价盐会导致反应逆转，并使可切割中间体与闭环松弛DNA之间的平衡分布向DNA重新封闭的方向转变。因此，表明盐影响切割反应但不影响重连反应。
