Skowron P, Kaczorowski T, Tucholski J, Podhajska A J
Department of Microbiology, University of Gdańsk, Poland.
Gene. 1993 Mar 15;125(1):1-10. doi: 10.1016/0378-1119(93)90738-o.
The DNA-binding properties of the FokI restriction endonuclease were studied using the gel-mobility-shift assay. Specific recognition of the cognate sequence and cleavage of DNA are distinguishable functions and can be separated. FokI binds to its recognition site predominantly as a monomer. At high concentrations, FokI exhibits a cooperative recognition sequence-dependent aggregation. In 20 mM KCl/10 mM Tris.HCl buffer, the binding constant of FokI to its cognate site is equal 6.0-7.9 x 10(8)/mol and is lower than the values for most gene-regulatory proteins. FokI binding is 600-1500 times weaker to non-cognate double-stranded DNA than to the GGATG site, and 30,000 times weaker to single-stranded DNA or tRNA. The method of Bading [Nucleic Acids Res. 16 (1988) 5241-5248], used for determining the stoichiometry of protein bound to DNA by gel-mobility-shift assay, is extended.
利用凝胶迁移率变动分析研究了FokI限制性内切核酸酶的DNA结合特性。对同源序列的特异性识别和DNA切割是可区分的功能，并且可以分开。FokI主要以单体形式结合到其识别位点。在高浓度下，FokI表现出协同的识别序列依赖性聚集。在20 mM KCl/10 mM Tris.HCl缓冲液中，FokI与其同源位点的结合常数等于6.0-7.9×10(8)/mol，低于大多数基因调节蛋白的值。FokI与非同源双链DNA的结合比与GGATG位点的结合弱600-1500倍，与单链DNA或tRNA的结合弱30,000倍。用于通过凝胶迁移率变动分析确定与DNA结合的蛋白质化学计量的Bading方法[《核酸研究》16(1988)5241-5248]得到了扩展。
