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首页> 外文期刊>Journal of Molecular Biology >DNA supercoiling during ATP-dependent DNA translocation by the type I restriction enzyme EcoAI.
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DNA supercoiling during ATP-dependent DNA translocation by the type I restriction enzyme EcoAI.

机译：I型限制酶EcoAI在ATP依赖性DNA转运过程中的DNA超螺旋。

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摘要

Type I restriction enzymes cleave DNA at non-specific sites far from their recognition sequence as a consequence of ATP-dependent DNA translocation past the enzyme. During this reaction, the enzyme remains bound to the recognition sequence and translocates DNA towards itself simultaneously from both directions, generating DNA loops, which appear to be supercoiled when visualised by electron microscopy. To further investigate the mechanism of DNA translocation by type I restriction enzymes, we have probed the reaction intermediates with DNA topoisomerases. A DNA cleavage-deficient mutant of EcoAI, which has normal DNA translocation and ATPase activities, was used in these DNA supercoiling assays. In the presence of eubacterial DNA topoisomerase I, which specifically removes negative supercoils, the EcoAI mutant introduced positive supercoils into relaxed plasmid DNA substrate in a reaction dependent on ATP hydrolysis. The same DNA supercoiling activity followed by DNA cleavage was observed with the wild-type EcoAI endonuclease. Positive supercoils were not seen when eubacterial DNA topoisomerase I was replaced by eukaryotic DNA topoisomerase I, which removes both positive and negative supercoils. Furthermore, addition of eukaryotic DNA topoisomerase I to the product of the supercoiling reaction resulted in its rapid relaxation. These results are consistent with a model in which EcoAI translocation along the helical path of closed circular DNA duplex simultaneously generates positive supercoils ahead and negative supercoils behind the moving complex in the contracting and expanding DNA loops, respectively. In addition, we show that the highly positively supercoiled DNA generated by the EcoAI mutant is cleaved by EcoAI wild-type endonuclease much more slowly than relaxed DNA. This suggests that the topological changes in the DNA substrate associated with DNA translocation by type I restriction enzymes do not appear to be the trigger for DNA cleavage. Copyright 2000 Academic Press.
机译：由于ATP依赖的DNA易位酶经过酶的作用，I型限制酶会在非特异性位点切割DNA，使其远离其识别序列。在该反应过程中，酶保持与识别序列的结合，并同时从两个方向向其自身转移DNA，生成DNA环，当通过电子显微镜观察时，DNA环似乎超螺旋。为了进一步研究I型限制酶使DNA易位的机制，我们用DNA拓扑异构酶探测了反应中间体。在这些DNA超螺旋分析中使用了具有正常DNA易位和ATPase活性的EcoAI的DNA切割缺陷型突变体。在能特异性去除阴性超螺旋的真细菌DNA拓扑异构酶I的存在下，EcoAI突变体在依赖ATP水解的反应中将阳性超螺旋引入到松弛的质粒DNA底物中。用野生型EcoAI核酸内切酶观察到相同的DNA超螺旋活性，随后进行DNA切割。当真核DNA拓扑异构酶I取代真细菌DNA拓扑异构酶I时，未见到阳性超螺旋，这消除了阳性和阴性超螺旋。此外，向超螺旋反应产物中加入真核DNA拓扑异构酶I导致其快速松弛。这些结果与一个模型一致，在该模型中，沿封闭的环状DNA双链体螺旋路径的EcoAI易位同时在收缩和扩展的DNA环中分别在移动复合体的前方生成了正超螺旋，在其后生成了负超螺旋。此外，我们显示，由EcoAI突变体产生的高度阳性超螺旋DNA被EcoAI野生型核酸内切酶裂解的速度比松弛DNA慢得多。这表明与通过I型限制酶进行的DNA易位相关的DNA底物的拓扑变化似乎不是DNA切割的触发因素。版权所有2000学术出版社。

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