DNA communications by Type III restriction endonucleases-confirmation of 1D translocation over 3D looping
DNA communications by Type III restriction endonucleases-confirmation of 1D translocation over 3D looping
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DOI：
10.1093/nar/gkh762
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发表时间：
2004-08-01
影响因子：
14.9
通讯作者：
Szczelkun, MD
中科院分区：
文献类型：
--
作者：
Peakman, LJ;Szczelkun, MD
III型限制性内切酶对DNA的切割受到DNA底物上识别位点的相对排列的严格控制——内切酶的活性通常只由头对头定向的序列触发。成千上万的碱基对可以将这些位点分开。在这种距离上的长距离通信可以通过一维（1D） DNA易位或三维DNA环来实现。为了区分这些选择，我们分析了EcoPI和EcoP15l在DNA链链上的活性，其中识别位点在相同或单独的环上。一对位点位于同一环上的底物可被有效地切割，而位于不同环上的链链则不能被切割。这些结果排除了简单的3D dna环活动。为了进一步表征相互作用，将EcoPI与携带两个识别位点的质粒孵育，其中两个识别位点穿插两个21res位点，通过Tn21分解进行位点特异性重组；抑制重组表明形成了稳定的DNA环。即使在可能发生EcoPI易位的情况下，也没有观察到抑制作用。
DNA cleavage by Type III restriction enzymes is governed strictly by the relative arrangement of recognition sites on a DNA substrate-endonuclease activity is usually only triggered by sequences in head-to-head orientation. Tens to thousands of base pairs can separate these sites. Long distance communication over such distances could occur by either one-dimensional (1D) DNA translocation or 3D DNA looping. To distinguish between these alternatives, we analysed the activity of EcoPI and EcoP15l on DNA catenanes in which the recognition sites were either on the same or separate rings. While substrates with a pair of sites located on the same ring were cleaved efficiently, catenanes with sites on separate rings were not cleaved. These results exclude a simple 3D DNA-looping activity. To characterize the interactions further, EcoPI was incubated with plasmids carrying two recognition sites interspersed with two 21res sites for site-specific recombination by Tn21 resolvase; inhibition of recombination would indicate the formation of stable DNA loops. No inhibition was observed, even under conditions where EcoPI translocation could also occur.