MECHANISM OF RESTRICTION ENDONUCLEASE ACTION

本项目的长期目标是了解 蛋白质对DNA的序列特异性识别。DNA识别 控制许多代谢活动，包括基因激活和 DNA的抑制、定点重组和起始 复制。了解这些蛋白质是如何工作的将提供 一种合理设计新蛋白质特性的框架 这可能会有医疗和药理学的应用。 研究的特殊蛋白质是嗜热性限制 核酸内切酶Tagi。提出了一种用于TAGI识别的模型 它的同源序列。对这一模式的进一步改进将 涉及野生型酶和突变体的特性 以及生化手段，分离具有改变的假定突变体 序列特异性、分离和鉴定 Tagi的异构体。 野生型核酸内切酶已经得到了过量生产和纯化 一个“正则位点划痕”突变体和一个“变构激活” 变种人。这些将被表征为序列特定结合， 使用各种解离、正则和“星形”位点裂解 DNA寡核苷酸和质粒底物。在协作中 在约翰·安德森博士的帮助下，提纯的Tagi将与 ITS同源寡核苷酸检测ITS三者 空间结构。 将使用两个密码子插入突变产生突变体， 寡核苷酸定向诱变和饱和诱变。 将开发两个活体筛查，可能有助于识别突变 具有改变的序列特异性。这些变种人应该会有帮助 定义序列特异性氢中涉及的氨基酸残基 连接到主槽，其他特定序列的触点， 变构激活的酶活性和蛋白质亚基 互动。 识别DNA的同裂异构体的性质和序列 与Tagi相似的结构主题将被确定。这个 同裂异体的氨基酸序列可能指向 序列特异性DNA所必需的TAGI中的功能同源性 承认。

The long term goal of this project is to understand the basis of sequence-specific recognition of DNA by proteins. DNA recognition governs many metabolic activities including gene activation and repression, site-specific recombination and initiation of DNA replication. Understanding how these proteins work will provide a framework for the rational design of new protein specificities that may have medical and pharmacological applications. The particular protein of study is the thermophilic restriction endonuclease TagI. A model has been proposed for TagI recognition of its cognate sequence. Further refinement of this model will involve characterization of wild type enzyme and mutants by genetic and biochemical means, isolation of putative mutants with altered sequence specificity, and isolation and characterization of an isoschizomer of TagI. Wild type endonuclease has been overproduced and purified as will a "canonical site nicking" mutant and an "allosteric activation" mutant. These will be characterized for sequence specific binding, dissociation, canonical and "star" site cleavage using a variety of DNA oligonucleotide and plasmid substrates. In collaboration with Dr. John Anderson, purified TagI will be co-crystallized with its cognate oligonucleotide for determination of its three dimensional structure. Mutants will be generated using two-codon insertion mutagenesis, oligonucleotide directed mutagenesis, and saturation mutagenesis. Two in vivo screens will be developed and may help identify mutants with altered sequence specificity. These mutants should help define amino acid residues involved in sequence-specific hydrogen bonds to the major groove, other sequence-specific contacts, allosteric activation of enzyme activity, and protein subunit interactions. The properties and sequence of an isoschizomer that recognizes DNA with similar structural motifs as TagI will be determined. The amino acid sequence of the isoschizomer might point to regions of functional homology in TagI necessary for sequence specific DNA recognition.