Selection of non-specific DNA cleavage sites by the type IC restriction endonuclease EcoR124I
Selection of non-specific DNA cleavage sites by the type IC restriction endonuclease EcoR124I
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DOI：
10.1006/jmbi.1997.1172
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发表时间：
1997-08-08
影响因子：
5.6
通讯作者：
Halford, SE
中科院分区：
文献类型：
--
作者：
Szczelkun, MD;Janscak, P;Halford, SE
IC型限制性内切酶EcoR124I特异性结合其识别序列，但随后以atp依赖机制将非特异性DNA通过复合体易位，因此该酶具有在远离识别位点的位点上切割DNA的潜力。我们仔细研究了在线性和圆形DNA底物上易位和切割之间的联系。在携带两个识别位点的线性DNA上，无论位点间的距离或相对方向如何，在距离识别位点较远的位点上，大多数切割发生在两个位点之间。在携带一个位点的环状DNA上，整个DNA发生了远端切割，而等效的线性分子在远端位点上发生的切割要少得多。这些结果与已发表的DNA黑客模型一致。然而，在所研究的每个分子上，在识别位点的+/-250 bp内也观察到离散的裂解位点。局部裂解不局限于特定的DNA序列，与DNA拓扑结构无关。我们提出了一个模型来解释远距离和局部裂解事件。在切割过程中挤出的DNA环的构象可能导致两个DNA片段靠近蛋白质上的限制性片段，一个靠近EcoR124I位点，另一个远离该位点：切割可能发生在任何一个片段上。另外，DNA靠近识别位点的切割可能是在任何广泛易位之前在扩展环中产生多个切口的结果。(C) 1997学术出版社有限公司
The Type IC restriction endonuclease EcoR124I binds specifically to its recognition sequence but subsequently translocates non-specific DNA past the complex in an ATP-dependent mechanism The enzyme thus has the potential to cleave DNA at loci distant from the recognition site. We have scrutinised the link between translocation and cleavage on linear and circular DNA substrates. On Linear DNA carrying two recognition sites, the majority of cleavages at loci distant from the recognition site occurred between the two sites, regardless of the inter-site distance or relative orientations. On circular DNA carrying one site, distant cleavages occurred throughout the DNA but an equivalent linear molecule underwent considerably fewer cleavages at distant loci. These results agree with published models for DNA hacking. However, on every molecule investigated, discrete cleavage sites were also observed within +/-250 bp of the recognition sites. The localised cleavages were not confined to particular DNA sequences and were independent of DNA topology. We propose a model to account for both distant and localised cleavage events. The conformation of the DNA loop extruded during hacking may result in two DNA segments being held in proximity to the restriction moiety on the protein, one close to the EcoR124I site and another distant from the site: cleavage may occur in either segment. Alternatively, the cutting of DNA close to recognition sites may be the result of multiple nicks being generated in the expanding loop before any extensive translocation. (C) 1997 Academic Press Limited.