Lee M P, Hsieh T
Department of Biochemistry, Duke University Medical Center, Durham, NC 27710.
Nucleic Acids Res. 1992 Oct 11;20(19):5027-33. doi: 10.1093/nar/20.19.5027.
Anti-tumor drug VM26 greatly stimulates topoisomerase II mediated DNA cleavage by stabilizing the cleavable complex. Addition of a strong detergent such as SDS to the cleavable complex induces the double stranded DNA cleavage. We demonstrate here that heat treatment can reverse the double stranded DNA cleavage; however, topoisomerase II remains bound to DNA even in the presence of SDS. This reversed complex has been shown to contain single strand DNA breaks with topoisomerase II covalently linked to the nicked DNA. Chelation of Mg++ by EDTA and the addition of salt to a high concentration also reverse the double strand DNA cleavage, and like heat reversion, topoisomerase II remains bound to DNA through single strand DNA break. The reversion complex can be analyzed and isolated by CsCl density gradient centrifugation. We have detected multiple discrete bands from such a gradient, corresponding to protein/DNA complexes with 1, 2, 3, ..... topoisomerase II molecules bound per DNA molecule. Analysis of topoisomerase II/DNA complexes isolated from the CsCl gradient indicates that there are single stranded DNA breaks associated with the CsCl stable complexes. Therefore, topoisomerase II/DNA complex formed in the presence of VM26 cannot be completely reversed to yield free DNA and enzyme. We discuss the possible significance of this finding to the mechanism of action of VM26 in the topoisomerase II reactions.
抗肿瘤药物VM26通过稳定可裂解复合物极大地刺激拓扑异构酶II介导的DNA裂解。向可裂解复合物中添加强去污剂如SDS会诱导双链DNA裂解。我们在此证明热处理可以逆转双链DNA裂解；然而，即使在存在SDS的情况下，拓扑异构酶II仍与DNA结合。已证明这种逆转复合物含有单链DNA断裂，拓扑异构酶II与带切口的DNA共价连接。EDTA螯合Mg++以及添加高浓度盐也能逆转双链DNA裂解，并且与热逆转一样，拓扑异构酶II通过单链DNA断裂仍与DNA结合。逆转复合物可以通过CsCl密度梯度离心进行分析和分离。我们从这样的梯度中检测到多个离散条带，对应于每个DNA分子结合有1、2、3……个拓扑异构酶II分子的蛋白质/DNA复合物。对从CsCl梯度中分离出的拓扑异构酶II/DNA复合物的分析表明，与CsCl稳定复合物相关存在单链DNA断裂。因此，在VM26存在下形成的拓扑异构酶II/DNA复合物不能完全逆转以产生游离的DNA和酶。我们讨论了这一发现对VM26在拓扑异构酶II反应中的作用机制的可能意义。
