Dreier J, MacWilliams M P, Bickle T A
Department of Microbiology, Biozentrum, Basel University, Switzerland.
J Mol Biol. 1996 Dec 13;264(4):722-33. doi: 10.1006/jmbi.1996.0672.
Type I restriction-modification systems bind to non-palindromic, bipartite recognition sequences. Although these enzymes methylate specific adenine residues within their recognition sequences, they cut DNA at sites up to several thousand base-pairs away. We have investigated the mechanism of how EcoR124II, a type IC restriction-modification system, selects the cleavage site. Restriction studies with different DNA constructs revealed that circular DNA requires only one non-methylated recognition sequence to be cut, whereas linear DNA needs at least two such sites. Cleavage of linear DNA is independent of site orientation. Further investigations of the linear substrates revealed a mechanism whereby the double-strand break is introduced between two recognition sequences. We propose a model for the selection of restriction sites by type I enzymes where two EcoR124II complexes bind to two recognition sequences. Lack of methylation at a site stimulates the enzyme to translocate DNA on both sides of the recognition sequence. Thus the two complexes approach each other and, at the point where they meet, they interact to introduce a double-strand break in the DNA.
I型限制-修饰系统与非回文的双组分识别序列结合。尽管这些酶会甲基化其识别序列内的特定腺嘌呤残基，但它们会在数千个碱基对以外的位点切割DNA。我们研究了I型C限制-修饰系统EcoR124II选择切割位点的机制。对不同DNA构建体的限制研究表明，环状DNA只需一个未甲基化的识别序列即可被切割，而线性DNA至少需要两个这样的位点。线性DNA的切割与位点方向无关。对线性底物的进一步研究揭示了一种机制，即双链断裂在两个识别序列之间引入。我们提出了一个I型酶选择限制位点的模型，其中两个EcoR124II复合物与两个识别序列结合。位点处甲基化的缺失刺激酶在识别序列两侧转运DNA。因此，两个复合物相互靠近，在它们相遇的点，它们相互作用以在DNA中引入双链断裂。
