DNA cleavage reactions by type II restriction enzymes that require two copies of their recognition sites
DNA cleavage reactions by type II restriction enzymes that require two copies of their recognition sites
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DOI：
10.1006/jmbi.2001.4892
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发表时间：
2001-08-17
影响因子：
5.6
通讯作者：
Halford, SE
中科院分区：
文献类型：
--
作者：
Embleton, ML;Siksnys, V;Halford, SE
几种II型限制性核酸内切酶在切割DNA之前与其靶序列的两个拷贝相互作用。三个这样的酶，NgoMIV，Cfr10I和NaeI，进行了测试的质粒与一个或两个副本的识别位点，并在catenanes含有两个相互连接的环的DNA与一个网站在每个环。这些酶表现出不同的行为模式。NgoMIV和NaeI裂解质粒的两个网站比一个网站和索烃在一个中间速率更快，而Cfr10I给所有三个基板上类似的稳态速率。Cfr 10 I和NgoMIV都将大部分具有两个位点的底物直接转化为在两个位点切割的产物，而NaeI一次仅切割一个位点。因此，所有三种酶在切割DNA之前通过三维空间使两个DNA位点突触。与Cfr10I和NgoMIV，这两个网站被切割在一个营业额，在与他们的四聚体结构一致的方式，而由NaeI的单一网站的裂解表明，第二个网站作为一个激活剂，而不是作为一个底物，如前所述。跨越两个站点的复合物具有更长的寿命与一个网站在每个环上的索烃比环状DNA上有两个网站，这表明索烃有更多的自由度，网站并置比质粒的顺式。(C)北京：科学出版社.
Several type II restriction endonucleases interact with two copies of their target sequence before they cleave DNA. Three such enzymes, NgoMIV, Cfr10I and NaeI, were tested on plasmids with one or two copies of their recognition sites, and on catenanes containing two interlinked rings of DNA with one site in each ring. The enzymes showed distinct patterns of behaviour. NgoMIV and NaeI cleaved the plasmid with two sites faster than that with one site and the catenanes at an intermediate rate, while Cfr10I gave similar steady-state rates on all three substrates. Both Cfr10I and NgoMIV converted the majority of the substrates with two sites directly to the products cut at both sites, while NaeI cleaved just one site at a time. All three enzymes thus synapse two DNA sites through three-dimensional space before cleaving DNA. With Cfr10I and NgoMIV, both sites are cleaved in one turnover, in a manner consistent with their tetrameric structures, while the cleavage of a single site by NaeI indicates that the second site acts not as a substrate but as an activator, as reported previously. The complexes spanning two sites have longer lifetimes on catenanes with one site in each ring than on circular DNA with two sites, which indicates that the catenanes have more freedom for site juxtaposition than plasmids with sites in cis. (C) 2001 Academic Press.