The DNA-proteins Interaction Unit, School of Biochemistry, University of Bristol, University Walk, Bristol BS8 1TD, UK.
The DNA-proteins Interaction Unit, School of Biochemistry, University of Bristol, University Walk, Bristol BS8 1TD, UK
Nucleic Acids Res. 2014 May;42(9):5809-22. doi: 10.1093/nar/gku187. Epub 2014 Mar 14.
Type II restriction-modification systems cleave and methylate DNA at specific sequences. However, the Type IIB systems look more like Type I than conventional Type II schemes as they employ the same protein for both restriction and modification and for DNA recognition. Several Type IIB proteins, including the archetype BcgI, are assemblies of two polypeptides: one with endonuclease and methyltransferase roles, another for DNA recognition. Conversely, some IIB proteins express all three functions from separate segments of a single polypeptide. This study analysed one such single-chain protein, TstI. Comparison with BcgI showed that the one- and the two-polypeptide systems differ markedly. Unlike the heterologous assembly of BcgI, TstI forms a homotetramer. The tetramer bridges two recognition sites before eventually cutting the DNA in both strands on both sides of the sites, but at each site the first double-strand break is made long before the second. In contrast, BcgI cuts all eight target bonds at two sites in a single step. TstI also differs from BcgI in either methylating or cleaving unmodified sites at similar rates. The site may thus be modified before TstI can make the second double-strand break. TstI MTase acts best at hemi-methylated sites.
II 型限制修饰系统在特定序列上切割和甲基化 DNA。然而，II 型 B 系统看起来更像 I 型而不是传统的 II 型方案，因为它们使用相同的蛋白质进行限制和修饰以及 DNA 识别。几种 II 型 B 蛋白，包括原型 BcgI，是由两条多肽组成的复合物：一条具有内切酶和甲基转移酶作用，另一条用于 DNA 识别。相反，一些 IIB 蛋白可以从单个多肽的不同片段表达所有三种功能。本研究分析了这样一种单链蛋白 TstI。与 BcgI 的比较表明，一单和两多肽系统差异显著。与 BcgI 的异源组装不同，TstI 形成同源四聚体。四聚体在最终切割位于两个识别位点两侧的两条链上的 DNA 之前桥接两个识别位点，但在每个位点，第一个双链断裂在第二个双链断裂之前很久就发生了。相比之下，BcgI 在单个步骤中切割两个位点的所有八个靶键。TstI 在甲基化或切割未修饰的位点方面也与 BcgI 不同，其速率相似。因此，在 TstI 能够进行第二个双链断裂之前，该位点可能已经被修饰。TstI MTase 在半甲基化位点上的活性最佳。
