MAPPING THE FUNCTIONAL DOMAINS OF AN ENDONUCLEASE

绘制核酸内切酶的功能域图

• 批准号: 2191036
• 负责人: MICHAEL D TOPAL
• 金额: $ 18.32万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-01-01 至 1998-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2191036
• 关键词: Actinomyces; DNA; endonuclease; gene mutation; genetic mapping; molecular biology; mutant; nucleic acid sequence; oligonucleotides; protein engineering; protein structure function; site directed mutagenesis

NaeI, a DNA endonuclease from an aerobic actinomycete, is a prototype for a new type of sequence-specific endonuclease that offers opportunities for new information about protein-DNA interactions and possibilities of new technologies. NaeI must bind an enhancer DNA element to cleave its recognition site (Fig.1): Enhancer and substrate elements are distinguished by the sequences immediately surrounding a core six-base recognition site that is required for binding. Enhancer induces assembly of an active cleavasome that involves conformational changes to the protein to activate catalysis. The characteristics of the NaeI-type enzymes and possible homology between one member, EcoRII, and the Int-family of recombinases indicates that this type of enzyme may be an evolutionary link between the restriction enzymes and the recombinases. The long-term objectives of this application are to use a protein engineering approach to alter the DNA specificity of NaeI endonuclease to recognize pathologic sequences in human DNA. Near term we will use molecular biology to map and characterize the functional domains of NaeI. Specific Aims include: 1) Mutate and select for different classes of NaeI mutants. 2) Select for mutations in the region required for NaeI dimerization. 3) Map the mutations to specific regions of the gene and determine the mutagenic changes to DNA sequence. 4) Site-directed mutagenesis of recognized functional domains. 5) Biochemical Characterization of the Mutations. 6) Change the specificity of NaeI-DNA recognition.

NaeI是一种来自好氧放线菌的DNA内切酶， 一种新型的序列特异性核酸内切酶， 关于蛋白质-DNA相互作用的新信息和新的可能性 技术. NaeI必须结合一个增强子DNA元件， 识别位点（图1）：增强子和底物元件是 通过直接围绕核心六个碱基的序列来区分 识别位点是结合所需的。 增强子诱导组装 涉及蛋白质构象变化的活性裂解体 来激活催化作用。 NaeI型酶的特性和 其中一个成员EcoRII和Int家族之间可能存在同源性， 重组酶的研究表明，这种类型的酶可能是一个进化的联系， 限制性内切酶和重组酶之间的联系。 本申请的长期目标是使用蛋白质 改变NaeI核酸内切酶的DNA特异性的工程方法， 识别人类DNA中的病理序列。 近期我们将使用 分子生物学来绘制和表征NaeI的功能结构域。 具体目标包括： 1)突变并选择不同种类的NaeI突变体。 2)选择NaeI二聚化所需区域中的突变。 3)将突变映射到基因的特定区域，并确定 DNA序列的突变变化。 4)识别功能域的定点诱变。 5)突变的生化表征。 6)改变NaeI-DNA识别的特异性。