Sutton C L, Mazumder A, Chen C H, Sigman D S
Department of Biological Chemistry, School of Medicine, University of California, Los Angeles 90024-1570.
Biochemistry. 1993 Apr 27;32(16):4225-30. doi: 10.1021/bi00067a009.
The Escherichia coli Trp repressor has been converted into an operator-specific nuclease by alkylating cysteine-49, inserted by site-directed mutagenesis, with 5-(iodoacetamido)-1,10-phenanthroline. In the presence of copper ion and thiol, high yields (> 50%) of double-stranded breaks of DNA are observed after a 20-min reaction. The high cleavage efficiency of this derivatized protein (Trp repressor E49C-OP) can be attributed to the proximity of cysteine-49 to the minor groove, the site of the C-1H of the deoxyribose and the target of the oxidative nuclease activity of (1,10-phenanthroline)copper. Since sequence position 49 is close to the protein's C2 dyad axis and adjacent to the minor groove, Trp repressor E49C-OP reacts with the operator DNA near the binding site of this symmetry locus of the protein. The patterns of scission of the trpR, aroH, and trpEDCBA operators (a) confirm the orientation of the repressor to the operator predicted from the X-ray study of a cocrystal (Otwinowski et al., 1988) and (b) support the model for tandem binding of the repressor to the trpR, aroH, and trpEDCBA operators based on DNase I footprinting and methylation interference (Kumamoto et al., 1987). There are one, two, and three binding sites for the repressor on the trpR, aroH, and trpEDCBA operators, respectively. In addition to providing a novel approach to studying the interactions of DNA binding proteins, 1,10-phenanthroline-derivatized proteins such as Trp repressor E49C-OP may be useful as rare cutters in the analysis of high molecular weight DNAs, especially if their binding specificities can be altered.
通过用5-(碘乙酰胺基)-1,10-菲咯啉对经定点诱变插入的半胱氨酸-49进行烷基化处理，已将大肠杆菌色氨酸阻遏物转化为一种序列特异性核酸酶。在铜离子和硫醇存在的情况下，20分钟反应后可观察到DNA双链断裂的高产率（>50%）。这种衍生化蛋白（色氨酸阻遏物E49C-OP）的高切割效率可归因于半胱氨酸-49靠近小沟、脱氧核糖C-1H的位置以及（1,10-菲咯啉）铜的氧化核酸酶活性的作用靶点。由于序列位置49靠近蛋白质的C2二元轴且与小沟相邻，色氨酸阻遏物E49C-OP在该蛋白质对称位点的结合位点附近与操纵基因DNA发生反应。trpR、aroH和trpEDCBA操纵基因（a）的断裂模式证实了从共晶体的X射线研究中预测的阻遏物与操纵基因的方向（奥特维诺夫斯基等人，1988年），并且（b）支持基于DNA酶I足迹法和甲基化干扰的阻遏物与trpR、aroH和trpEDCBA操纵基因串联结合的模型（熊本等人，1987年）。阻遏物在trpR、aroH和trpEDCBA操纵基因上分别有一个、两个和三个结合位点。除了提供一种研究DNA结合蛋白相互作用的新方法外，1,10-菲咯啉衍生化的蛋白，如色氨酸阻遏物E49C-OP，在高分子量DNA分析中作为稀有切割酶可能是有用的，特别是如果它们的结合特异性能够改变的话。
