SITE-SPECIFIC RECOMBINATION

位点特异性重组

• 批准号: 2175257
• 负责人: JEFFREY F GARDNER
• 金额: $ 24.37万
• 依托单位: UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
• 依托单位国家: 美国
• 财政年份: 1980
• 资助国家: 美国
• 起止时间: 1980-12-01 至 1996-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2175257
• 关键词: DNA binding protein; DNA footprinting; bacteriophage lambda; binding proteins; developmental genetics; enzyme activity; gene expression; gene mutation; genetic mapping; genetic recombination; integrase; microorganism genetics; molecular cloning; mutant; nucleic acid sequence; polymerase chain reaction; radiotracer; site directed mutagenesis; western blottings

Site-specific recombination systems are important in controlling development and gene expression in a diverse array of organisms ranging from bacteria to humans. The long range goal of this project is to understand how bacteriophage lambda carries-out site-specific recombination. Both biochemical and genetic approaches will be used to characterize the protein-protein and protein-DNA interactions that occur during the assembly of recombination complexes (intasomes) and the process of strand exchange. The phage-encoded integrase (Int) protein participates in intasome formation and catalyzes strand exchange. Mutants with altered DNA binding specificities will be isolated to determine which amino acid residues in Int are responsible for DNA recognition. Other Int mutants will be isolated and characterized in biochemical assays that will determine the defects of individual mutant proteins in the recombination pathway. The host-encoded integration host factor (IHF) participates in intasome formation by inducing bends in the DNA. A combination of in vivo and in vitro mutagenesis approaches will be used to isolate and to characterize altered DNA binding specificity mutants of IHF. Such mutants will identify the amino acid residues in IHF that interact with DNA and will be useful in interpreting structures derived from physical studies. The phage-encoded excisionase (Xis) and the host-encoded factor for inversion stimulation (FIS) promoter excisive recombination. Xis interacts cooperatively with Int and FIS. Xis mutants will be isolated that are defective in cooperative interactions with Int and FIS and the mutant proteins will be used to determine the mechanism(s) of the cooperative interactions.

特定部位的重组系统在控制 不同生物体的发育和基因表达 从细菌到人类。这个项目的长期目标是 了解噬菌体lambda是如何进行特定部位的 重组。生化和遗传方法都将用于 描述发生的蛋白质-蛋白质和蛋白质-DNA相互作用 在重组复合体(内含体)和 链交换的过程。噬菌体编码整合酶(Int)蛋白 参与内切酶的形成并催化链交换。 DNA结合特性改变的突变体将被分离出来 确定Int中哪些氨基酸残基对DNA负责 承认。其他Int突变体将被分离并在 将确定单个突变体缺陷的生化分析 重组途径中的蛋白质。主机编码的集成主机 IHF因子通过诱导肠系膜细胞的弯曲参与肠套的形成。 DNA体内和体外诱变方法的结合将 用于分离和鉴定改变的DNA结合特异性 IHF的突变体。这样的突变体将识别氨基酸残基 与DNA相互作用的IHF将在解释结构中有用 源自物理研究。噬菌体编码的摘除酶(XIS)和 宿主编码的逆转刺激因子(FIS)启动子激动子 重组。XIS与Int和FIS协同交互。西雅图 在合作作用中有缺陷的突变体将被隔离 利用Int和FIS，突变蛋白将被用来确定 合作互动机制(S)。