Nobbs T J, Szczelkun M D, Wentzell L M, Halford S E
Department of Biochemistry, School of Medical Sciences, Bristol, BS8 1TD, UK.
J Mol Biol. 1998 Aug 21;281(3):419-32. doi: 10.1006/jmbi.1998.1966.
A mechanism for the precise excision of DNA between two target sites was elucidated by analysing the individual steps during the reactions of the SfiI endonuclease on a plasmid with two SfiI sites. Previous studies had indicated that SfiI is a tetrameric protein that binds to two copies of its recognition site before cleaving both sites in both strands. In this study, the concerted cleavage of four phosphodiester bonds was shown to arise from four consecutive reactions that had similar values for their intrinsic rate constants. Each reaction is presumably mediated by one of the four active sites in the tetramer and all four were generally completed within the life-time of the complex between the protein and two recognition sites, though products cleaved in one or two phosphodiester bonds were also detected following premature dissociation of the enzyme-substrate complex at elevated temperatures. At the physiological temperature for this enzyme, all four bonds were cleaved within one minute but the subsequent dissociation of the enzyme-product complex, liberating the excised segment of DNA, took about one hour. The tetrameric structure for SfiI was confirmed by equilibrium centrifugation.
通过分析SfiI核酸内切酶在具有两个SfiI位点的质粒上反应过程中的各个步骤，阐明了在两个靶位点之间精确切除DNA的机制。先前的研究表明，SfiI是一种四聚体蛋白，在切割两条链上的两个位点之前，它会结合到其识别位点的两个拷贝上。在这项研究中，四个磷酸二酯键的协同切割被证明是由四个连续反应产生的，这些反应的内在速率常数具有相似的值。每个反应大概由四聚体中的四个活性位点之一介导，尽管在高温下酶-底物复合物过早解离后也检测到了切割一个或两个磷酸二酯键的产物，但所有四个反应通常在蛋白质与两个识别位点之间的复合物寿命内完成。在该酶的生理温度下，所有四个键在一分钟内被切割，但随后酶-产物复合物的解离，释放出切除的DNA片段，大约需要一小时。通过平衡离心法证实了SfiI的四聚体结构。
