Meller V H, Fisher P A
Department of Pharmacological Sciences, State University of New York at Stony Brook 11794-8651, USA.
J Cell Sci. 1995 Apr;108 ( Pt 4):1651-7. doi: 10.1242/jcs.108.4.1651.
The nuclear distribution of Drosophila DNA topoisomerase II was determined by immunoblot analysis after nuclease digestion and cell fractionation. About 60% of DNA topoisomerase II could be removed from nuclei by RNase A, about 70% by DNase I, and about 90% by incubation with both enzymes together or with micrococcal nuclease. Nuclease treatment of nuclei did not affect the distribution of lamins Dm1 and Dm2 or other nuclear proteins similarly. Nuclease-mediated solubilization of DNA topoisomerase II from Drosophila nuclei was also dependent on NaCl concentration. Solubilization was not efficient below 100 mM NaCl. Sucrose velocity gradient ultracentrifugation demonstrated that DNA topoisomerase II solubilized from nuclei by either RNase A or DNase I migrated at about 9 S, as expected for the homodimer. Results of chemical crosslinking supported this observation. We conclude that DNA topoisomerase II has both RNA- and DNA-dependent anchorages in Drosophila embryo nuclei.
通过核酸酶消化和细胞分级分离后进行免疫印迹分析，确定了果蝇DNA拓扑异构酶II的核分布。约60%的DNA拓扑异构酶II可被核糖核酸酶A从细胞核中去除，约70%可被脱氧核糖核酸酶I去除，约90%可通过与两种酶一起孵育或与微球菌核酸酶孵育去除。对细胞核进行核酸酶处理对核纤层蛋白Dm1和Dm2或其他核蛋白的分布没有类似影响。核酸酶介导的从果蝇细胞核中溶解DNA拓扑异构酶II也依赖于NaCl浓度。在100 mM NaCl以下，溶解效率不高。蔗糖速度梯度超速离心表明，通过核糖核酸酶A或脱氧核糖核酸酶I从细胞核中溶解的DNA拓扑异构酶II以约9 S的速度迁移，这与同型二聚体的预期一致。化学交联结果支持了这一观察。我们得出结论，DNA拓扑异构酶II在果蝇胚胎细胞核中具有RNA和DNA依赖性的锚定作用。
