Faculty of Bioengineering and Bioinformatics, Chemistry Department, and Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Leninskie Gory 1, 119991 Moscow, Russia.
Analyst. 2011 Mar 21;136(6):1227-33. doi: 10.1039/c0an00545b. Epub 2011 Jan 28.
The interaction of (cytosine-5)-DNA methyltransferase SsoII (M.SsoII) with double-stranded DNA was studied by means of thickness shear mode acoustic method (TSM) and gel electrophoresis. M.SsoII recognizes in double-stranded DNA the methylation site 5'-CCNGG-3' (N=A, C, G, T) and methylates the inner cytosine residue. M.SsoII also acts as a transcription factor via binding to the regulatory site 5'-AGGACAAATTGTCCT-3' in the promoter region of SsoII restriction-modification system. We designed three 60-mer biotinylated DNA duplexes: with the methylation site (60met), with the regulatory site (60reg), and without a specific binding site (60oct). A strong binding of M.SsoII with each one of the studied DNA immobilized on the TSM transducer has been shown. The equilibrium dissociation constants, K(D), of the M.SsoII-DNA complexes decreased in the order 60oct>60reg>60met, suggesting a higher stability of M.SsoII-60met complex in comparison with the others. The association rate constant, k(a), was also higher for 60met, while similar values were obtained for 60reg and 60oct. The difference in the kinetic parameters for 60met and 60reg suggested a possible way of coordination between the two M.SsoII functions in a cell.
通过厚度剪切模式声学方法（TSM）和凝胶电泳研究了（胞嘧啶-5）-DNA 甲基转移酶 SsoII（M.SsoII）与双链 DNA 的相互作用。M.SsoII 在双链 DNA 中识别甲基化位点 5'-CCNGG-3'（N=A、C、G、T）并甲基化内部胞嘧啶残基。M.SsoII 还通过结合 SsoII 限制修饰系统启动子区域的调节位点 5'-AGGACAAATTGTCCT-3'作为转录因子发挥作用。我们设计了三个 60 个碱基对的生物素标记 DNA 双链体：带有甲基化位点（60met）、带有调节位点（60reg）和没有特定结合位点（60oct）。研究表明，固定在 TSM 换能器上的每个研究 DNA 与 M.SsoII 均具有强烈的结合。M.SsoII-DNA 复合物的平衡解离常数 K(D)按 60oct>60reg>60met 的顺序降低，表明与其他复合物相比，M.SsoII-60met 复合物更稳定。结合速率常数 k(a)对于 60met 也更高，而 60reg 和 60oct 则获得相似的值。60met 和 60reg 的动力学参数差异表明，在细胞中两种 M.SsoII 功能之间可能存在协调方式。
