SEQUENCE-SPECIFIC DNA PROTEIN INTERACTIONS

我们建议利用EcoRI之间的高度特异性交互作用 含有序列d(GAATTC)的核酸内切酶和寡核苷酸 序列特异性DNA-蛋白质识别研究的模型系统。 这项研究的主要目的是剖析大脑的整体功能 将核酸内切酶转化为可操作定义的组分活动；然后 使用这些数据来制定详细的功能描述 该限制性内切酶与其同源DNA之间的相互作用。我们有 说明了这种功能分解可以通过使用 酶的突变或蛋白水解性改变形式。在这里我们 建议通过调查四个具体领域在此基础上再接再厉： A)确定对结合功能至关重要的氨基酸残基 通过化学修饰技术；b)通过化学方法识别 修饰，分子中参与的区域 DNA结合时的构象迁移率；c)询问 失去DNA切割能力的胰酶核心内切酶仍保留 完整酶的全序列特异性以及是否 DNA-蛋白质相互作用的静电成分已经改变；d) 鉴定由突变的核酸内切酶形成的DNA磷酸接触 减少与DNA的静电相互作用。

We propose to use the highly specific interaction between EcoRI endonuclease and oligonucleotides containing the sequence d(GAATTC) as a model system for the study of sequence specific DNA-protein recognition. The principal goals of this research are to dissect the overall function of the endonuclease into operationally defined component activities; and then to use these data to develop a detailed functional description of the interaction between this restriction enzyme and its cognate DNA. We have shown that this functional dissection can be achieved through the use of mutationally or proteolytically altered forms of the enzyme. Here we propose to build on this foundation by investigating four specific areas: a) to identify amino acid residues which are essential for binding function by chemical modification techniques; b) to identify, by chemical modification, the regions of the molecule which participate in conformational mobility upon DNA binding; c) to ask whether or not a tryptic core endonuclease which has lost DNA cleavage ability retains the full sequence specificity of intact enzyme and also whether or not the electrostatic component of the DNA-protein interaction has been altered; d) to identify DNA phosphate contacts made by a mutant endonuclease having decreased electrostatic interaction with DNA.