Szczelkun M D, Dillingham M S, Janscak P, Firman K, Halford S E
Department of Biochemistry, Centre for Molecular Recognition, University of Bristol, UK.
EMBO J. 1996 Nov 15;15(22):6335-47.
Type I restriction endonucleases such as EcoR124I cleave DNA at undefined loci, distant from their recognition sequences, by a mechanism that involves the enzyme tracking along the DNA between recognition and cleavage sites. This mechanism was examined on plasmids that carried recognition sites for EcoR124I and recombination sites for resolvase, the latter to create DNA catenanes. Supercoiled substrates with either one or two restriction sites were linearized by EcoR124I at similar rates, although the two-site molecule underwent further cleavage more readily than the one-site DNA. The catenane from the plasmid with one EcoR124I site, carrying the site on the smaller of the two rings, was cleaved by EcoR124I exclusively in the small ring, and this underwent multiple cleavage akin to the two-site plasmid. Linear substrates derived from the plasmids were cleaved by EcoR124I at very slow rates. The communication between recognition and cleavage sites therefore cannot stem from random looping. Instead, it must follow the DNA contour between the sites. On a circular DNA, the translocation of non-specific DNA past the specifically bound protein should increase negative supercoiling in one domain and decrease it in the other. The ensuing topological barrier may be the trigger for DNA cleavage.
I型限制性内切酶，如EcoR124I，通过一种机制在远离其识别序列的未定义位点切割DNA，该机制涉及酶在识别位点和切割位点之间沿着DNA追踪。在携带EcoR124I识别位点和重组酶重组位点的质粒上研究了这种机制，后者用于产生DNA连环体。具有一个或两个限制位点的超螺旋底物被EcoR124I以相似的速率线性化，尽管具有两个位点的分子比具有一个位点的DNA更容易进行进一步切割。来自具有一个EcoR124I位点的质粒的连环体，该位点位于两个环中较小的环上，被EcoR124I仅在小环中切割，并且这经历了类似于具有两个位点的质粒的多次切割。源自质粒的线性底物被EcoR124I以非常慢的速率切割。因此，识别位点和切割位点之间的通讯不可能源于随机环化。相反，它必须沿着位点之间的DNA轮廓进行。在环状DNA上，非特异性DNA经过特异性结合蛋白的易位应该会增加一个结构域中的负超螺旋并降低另一个结构域中的负超螺旋。由此产生的拓扑屏障可能是DNA切割的触发因素。
