ECORI FUNCTION ASSESSED BY SITE-DIRECTED MUTAGENESIS

我们建议协调结构，生物化学和遗传学研究的 EcoRI内切酶，以评估结构的关系， 这种蛋白质的功能机制。 具体目标是 研究建议是将定义的突变引入EcoRI 核酸内切酶基因，突变核酸内切酶的分析，和掺入 这些分析的结果转化为一个工作结构模型， EcoRI内切酶-底物相互作用。 突变研究最初 将集中于内切核酸酶的40个氨基酸区段，其至少 在某种程度上，已被确定与DNA直接接触， 衬底 该区域还包括已被修饰的氨基酸序列。 纳入计算机图形开发的工作模型， 识别EcoRI独特的六碱基对DNA底物 核酸内切酶。 G大于A且C大于T过渡 突变将被限制在基因组的指定区域， 选择的亚硫酸氢盐诱变的适当的“缺失环”， 异源双链质粒分子。 其他的突变将被引入这个 区域通过引物错配修复合成，并最终通过 开发“编码盒”， 突变改变。 核酸内切酶变体，由核苷酸定义 序列测定，将分析许多功能 参数，例如，二聚体结构稳定性，热稳定性，动力学 DNA水解，EcoRI* 活性，识别特性，结合 平衡研究、与DNA底物的接触点和评估 静电和非静电的相互作用。 的结果予以 分析将参考野生型内切核酸酶和 预期的EcoRI内切核酸酶底物的结构解析 完善EcoRI各个步骤的工作模型的复杂性 内切酶机制 后一项工作将利用计算机图形 设施在加州大学旧金山分校和计算机图形建模方法将 将其纳入特定突变改变的设计中， EcoRi核酸内切酶分子机制的不断完善 与底物反应。

We propose to coordinate structural, biochemical and genetic studies of the EcoRI endonuclease in order to assess the relationship of the structure of this protein to its functional mechanism. The specific aims of this research proposal are the introduction of defined mutations into the EcoRI endonuclease gene, analysis of the mutant endonucleases, and incorporation of the results of these analyses into a working structural model of the EcoRI endonuclease-substrate interaction. The mutational studies initially will focus on a 40 amino acid segment of the endonuclease which, at least in part, has been determined to be in direct contact with the DNA substrate. This region also includes an amino acid sequence which has been incorporated into a computergraphic developed working model for the recognition of the unique six-base pair DNA substrate of the EcoRI endonuclease. G greater than A and C greater than T transitional mutatations will be limited to the indicated region of the genome by selected bisulfite mutagenesis of the appropriate "deletion loop" of heteroduplex plasmid molecules. Other mutations will be introduced in this region by primer mismatched repair synthesis, and eventually by the development of "coding cassettes" for completely flexible designed mutational alterations. The endonuclease variants, define by nucleotide sequence determinations, will be analyzed for numerous functional parameters, e.g., stability of dimer structure, thermostability, kinetics of DNA hydrolysis, EcoRI* activity, recognition properties, binding equilibrium studies, points of contacts with DNA substrate, and assessment of electrostatic and non-electrostatic interactions. The results of these analyses will be referenced to the wild type endonuclease and the anticipated structural resolution of the EcoRI endonuclease-substrate complex to refine working models of the various steps of the EcoRI endonuclease mechanism. The latter effort will utilize the computergraphic facility at UCSF and the computergraphic modeling approach will be incorporated into the designing of specific mutational alterations for continued refinement of the molecular mechanism of the EcoRi endonuclease reaction with its substrate.