ATP 依赖性 DNA 螺旋追踪过程中的模板超螺旋：猿猴病毒 40 大肿瘤抗原的研究
Template supercoiling during ATP-dependent DNA helix tracking: studies with simian virus 40 large tumor antigen.
作者信息
Yang L, Jessee C B, Lau K, Zhang H, Liu L F
机构信息
Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD 21205.
出版信息
Proc Natl Acad Sci U S A. 1989 Aug;86(16):6121-5. doi: 10.1073/pnas.86.16.6121.
Incubation of topologically relaxed plasmid DNA with simian virus 40 (SV40) large tumor antigen (T antigen), ATP, and eubacterial DNA topoisomerase I resulted in the formation of highly positively supercoiled DNA. Eukaryotic DNA topoisomerase I could not substitute for eubacterial DNA topoisomerase 1 in this reaction. Furthermore, the addition of eukaryotic topoisomerase I to a preincubated reaction mixture containing both T antigen and eubacterial topoisomerase I caused rapid relaxation of the positively supercoiled DNA. These results suggest that SV40 T antigen can introduce topoisomerase-relaxable supercoils into DNA in a reaction coupled to ATP hydrolysis. We interpret the observed T antigen supercoiling reaction in terms of a recently proposed twin-supercoiled-domain model that describes the mechanics of DNA helix-tracking processes. According to this model positive and negative supercoils are generated ahead of and behind the moving SV40 T antigen, respectively. The preferential relaxation of negative supercoils by eubacterial DNA topoisomerase I explains the accumulation of positive supercoils in the DNA template. The supercoiling assay using DNA conformation-specific eubacterial DNA topoisomerase I may be of general use for the detection of ATP-dependent DNA helix-tracking proteins.
将拓扑松弛的质粒DNA与猿猴病毒40（SV40）大肿瘤抗原（T抗原）、ATP和真细菌DNA拓扑异构酶I一起温育，导致形成高度正超螺旋DNA。在该反应中，真核DNA拓扑异构酶I不能替代真细菌DNA拓扑异构酶I。此外，将真核拓扑异构酶I添加到预先温育的含有T抗原和真细菌拓扑异构酶I的反应混合物中，会使正超螺旋DNA迅速松弛。这些结果表明，SV40 T抗原可以在与ATP水解偶联的反应中，将拓扑异构酶可松弛的超螺旋引入DNA。我们根据最近提出的双超螺旋结构域模型来解释观察到的T抗原超螺旋反应，该模型描述了DNA螺旋追踪过程的机制。根据这个模型，正超螺旋和负超螺旋分别在移动的SV40 T抗原之前和之后产生。真细菌DNA拓扑异构酶I对负超螺旋的优先松弛解释了DNA模板中正超螺旋的积累。使用DNA构象特异性真细菌DNA拓扑异构酶I的超螺旋测定法可能普遍用于检测ATP依赖性DNA螺旋追踪蛋白。
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