SEQUENCE-SPECIFIC DNA PROTEIN INTERACTIONS

我们建议利用EcoRI之间的高度特异性交互作用 核酸内切酶和含有正常核苷酸的寡核苷酸 (GAATTC)或变体识别站点作为模型系统 序列特异性DNA-蛋白质相互作用的研究。我们有 展示了静电相互作用的意外作用 DNA裂解中的GAATpTC。我们使用了关于均衡的数据 结合和切割动力学来制定一个精炼的模型，它 中碱基替换的效果进行正确排序。 识别站点。我们还展示了接触 在蛋白质和特定的DNA磷酸之间发生变化 当该酶结合到包含单个 碱基取代，以及磷酸盐在pXGAATTC上的接触 取决于侧翼序列。在这里，我们建议建立 在此基础上：a)确定是否以及如何 氢键与识别位上碱基的相互作用 相互依赖；b)评估职能作用和 蛋白质与蛋白质相互作用的相互依赖关系 个体DNA磷酸盐；c)系统地研究 侧翼碱基序列对磷酸盐影响的依据 相互作用；以及d)鉴定其化学成分为 修饰会使核酸内切酶失活。

We propose to use the highly specific interaction between EcoRI endonuclease and oligonucleotides containing the normal (GAATTC) or variant recognition sites as a model system for the study of sequence-specific DNA-protein interactions. We have demonstrated an unexpected role for electrostatic interactions at GAATpTC in DNA cleavage. We have used data on equilibrium binding and cleavage kinetics to formulate a refined model which correctly orders the effects of base substitutions in the recognition site. We have also demonstrated that contacts between the protein and particular DNA phosphates are altered when the enzyme binds to recognition sites containing a single base substitution, and that the phosphate contact at pXGAATTC is dependent upon flanking sequence. Here we propose to build upon this foundation by: a) Determining whether and how hydrogen bonding interactions to bases in the recognition site depend upon each other; b) Assessing the functional roles and interdependences of interactions between the protein and individual DNA phosphates; c) Systematically investigating the basis for the influence of flanking base sequence on phosphate interactions; and d) Identifying a carboxyl residue whose chemical modification inactivates the endonuclease.