DNA环化和易位为III型限制酶提供了一种最佳的切割机制。
DNA looping and translocation provide an optimal cleavage mechanism for the type III restriction enzymes.
作者信息
Crampton Neal, Roes Stefanie, Dryden David T F, Rao Desirazu N, Edwardson J Michael, Henderson Robert M
机构信息
Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge, UK.
出版信息
EMBO J. 2007 Aug 22;26(16):3815-25. doi: 10.1038/sj.emboj.7601807. Epub 2007 Jul 26.
Abstract
EcoP15I is a type III restriction enzyme that requires two recognition sites in a defined orientation separated by up to 3.5 kbp to efficiently cleave DNA. The mechanism through which site-bound EcoP15I enzymes communicate between the two sites is unclear. Here, we use atomic force microscopy to study EcoP15I-DNA pre-cleavage complexes. From the number and size distribution of loops formed, we conclude that the loops observed do not result from translocation, but are instead formed by a contact between site-bound EcoP15I and a nonspecific region of DNA. This conclusion is confirmed by a theoretical polymer model. It is further shown that translocation must play some role, because when translocation is blocked by a Lac repressor protein, DNA cleavage is similarly blocked. On the basis of these results, we present a model for restriction by type III restriction enzymes and highlight the similarities between this and other classes of restriction enzymes.
摘要
EcoP15I是一种III型限制酶，它需要两个特定方向的识别位点，两个位点之间的距离可达3.5千碱基对，才能有效切割DNA。位点结合的EcoP15I酶在两个位点之间进行通讯的机制尚不清楚。在这里，我们使用原子力显微镜来研究EcoP15I-DNA预切割复合物。从形成的环的数量和大小分布来看，我们得出结论，观察到的环不是由易位产生的，而是由位点结合的EcoP15I与DNA的非特异性区域之间的接触形成的。这一结论得到了理论聚合物模型的证实。进一步表明易位必定起了一定作用，因为当易位被乳糖阻遏蛋白阻断时，DNA切割同样被阻断。基于这些结果，我们提出了一个III型限制酶限制作用的模型，并强调了它与其他类限制酶之间的相似性。
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