SEQUENCE-SPECIFIC DNA PROTEIN INTERACTIONS

我们建议利用EcoRI之间的高度特异性相互作用 核酸内切酶和含有序列d（GAATTC）作为核酸内切酶的寡核苷酸。 用于序列特异性DNA-蛋白质识别研究的模型系统。 本研究的主要目的是剖析 将核酸内切酶转化为操作上定义的组分活性;然后 使用这些数据来开发详细的功能描述， 这种限制性内切酶和它的同源DNA之间的相互作用。 我们有 表明这种功能性解剖可以通过使用 酶的突变或蛋白水解改变形式。 这里我们 建议在这一基础上，调查四个具体领域： a）鉴定结合功能所必需的氨基酸残基 通过化学改性技术; B）通过化学改性技术 修饰，参与修饰的分子区域 DNA结合时的构象迁移率; c）询问是否存在 失去DNA切割能力的胰蛋白酶核心核酸内切酶保留了 完整酶的完整序列特异性，以及 DNA-蛋白质相互作用的静电分量已经改变; d） 为了鉴定由突变核酸内切酶产生的DNA磷酸接触，所述突变核酸内切酶具有 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.