Abrosimova Liudmila A, Kubareva Elena A, Migur Anzhela Yu, Gavshina Aleksandra V, Ryazanova Aleksandra Yu, Norkin Maxim V, Perevyazova Tatiana A, Wende Wolfgang, Hianik Tibor, Zheleznaya Liudmila A, Oretskaya Tatiana S
Chemistry Department and A.N. Belozersky Institute of Physico-Chemical Biology, M.V. Lomonosov Moscow State University, Leninskye gory 1, 119991 Moscow, Russia.
Chemistry Department and A.N. Belozersky Institute of Physico-Chemical Biology, M.V. Lomonosov Moscow State University, Leninskye gory 1, 119991 Moscow, Russia; Institute of Molecular Biology GmbH, Ackermannweg 4, 55128 Mainz, Germany.
Biochim Biophys Acta. 2016 Sep;1864(9):1072-1082. doi: 10.1016/j.bbapap.2016.05.008. Epub 2016 May 20.
Nicking endonucleases are enzymes that recognize specific sites in double-stranded DNA and cleave only one strand at a predetermined position. These enzymes are involved in DNA replication and repair; they can also function as subunits of bacterial heterodimeric restriction endonucleases. One example of such a proteins is the restriction endonuclease BspD6I (R.BspD6I) from Bacillus species strain D6, which consists of the large subunit - nicking endonuclease BspD6I (Nt.BspD6I), and the small subunit (ss.BspD6I). Nt.BspD6I can function independently. Similar enzymes are now widely used in numerous biotechnological applications. The aim of this study was to investigate the fundamental properties of two subunits of R.BspD6I and their interdependence in the course of R.BspD6I activity.
The binding and hydrolysis of DNA duplexes by R.BspD6I are primary analyzed by gel electrophoresis. To elucidate the difference between Nt.BspD6I interaction with the substrate and product of hydrolysis, the thickness shear mode acoustic method is used.
The thermodynamic and kinetic parameters of the Nt.BspD6I interaction with DNA are determined. For the first time we demonstrated that Nt.BspD6I bends the DNA during complex formation. Nt.BspD6I is able to form complexes with the product nicked in the top strand and ss.BspD6I cleaves the bottom strand of the DNA consecutively. Furthermore, the influence of dA methylation in the R.BspD6I recognition site on ss.BspD6I activity is analyzed.
The obtained results provide evidence that Nt.BspD6I coordinates the activity of R.BspD6I by strictly coupling of the bottom strand cleavage by ss.BspD6I to the top strand cleavage.
切口核酸酶是一类能识别双链DNA中的特定位点并仅在预定位置切割一条链的酶。这些酶参与DNA复制和修复；它们还可作为细菌异源二聚体限制性核酸内切酶的亚基发挥作用。此类蛋白质的一个例子是来自芽孢杆菌属菌株D6的限制性核酸内切酶BspD6I（R.BspD6I），它由大亚基——切口核酸酶BspD6I（Nt.BspD6I）和小亚基（ss.BspD6I）组成。Nt.BspD6I能够独立发挥作用。类似的酶如今广泛应用于众多生物技术领域。本研究的目的是探究R.BspD6I两个亚基的基本特性以及它们在R.BspD6I活性过程中的相互依赖性。
通过凝胶电泳初步分析R.BspD6I对DNA双链的结合和水解情况。为阐明Nt.BspD6I与水解底物及产物相互作用的差异，采用厚度剪切模式声学方法。
确定了Nt.BspD6I与DNA相互作用的热力学和动力学参数。我们首次证明Nt.BspD6I在复合物形成过程中会使DNA弯曲。Nt.BspD6I能够与顶部链切口的产物形成复合物，且ss.BspD6I会依次切割DNA的底部链。此外，还分析了R.BspD6I识别位点中dA甲基化对ss.BspD6I活性的影响。
所获结果证明Nt.BspD6I通过将ss.BspD6I对底部链的切割与顶部链的切割严格偶联来协调R.BspD6I的活性。
