Yonezawa A, Sugiura Y
Institute for Chemical Research, Kyoto University, Japan.
Biochim Biophys Acta. 1994 Oct 18;1219(2):369-79. doi: 10.1016/0167-4781(94)90061-2.
We investigate the interaction of FokI with its DNA recognition sequence by several footprinting techniques. Methylation of three guanine bases in the recognition sequence 5'-GGATG-3' is strongly protected by FokI binding, whereas other guanine bases are not masked from the modification. In footprinting using the methidiumpropyl-EDTA-Fe(II) complex, binding of FokI strongly inhibits cleavage by the footprinting reagent at and near the recognition sequence. In high-resolution footprinting techniques using hydroxyl radical and the bleomycin-Fe(II) complex, all footprints in each binding site clearly face one side of the DNA helix. Interference analysis with FokI digestion by preethylation of phosphate groups suggests that essential phosphates for FokI digestion are located at and near the recognition sequence and the cleavage site. Evidently, the results indicate that (i) the sequence-recognition of FokI occurs in the major groove and that (ii) the enzyme interacts with its target DNA from one side of the DNA helix.
我们通过几种足迹分析技术研究了FokI与其DNA识别序列的相互作用。在识别序列5'-GGATG-3'中，三个鸟嘌呤碱基的甲基化受到FokI结合的强烈保护，而其他鸟嘌呤碱基则不会被修饰所掩盖。在使用甲基丙基乙二胺四乙酸铁（II）复合物进行的足迹分析中，FokI的结合强烈抑制了足迹试剂在识别序列及其附近的切割。在使用羟基自由基和博来霉素-铁（II）复合物的高分辨率足迹分析技术中，每个结合位点的所有足迹都明显朝向DNA螺旋的一侧。通过磷酸基团的预乙基化对FokI消化进行的干扰分析表明，FokI消化所需的关键磷酸基团位于识别序列和切割位点及其附近。显然，结果表明：（i）FokI的序列识别发生在大沟中，并且（ii）该酶从DNA螺旋的一侧与其靶DNA相互作用。
