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.

位点特异性重组系统在控制 发展和基因表达在各种各样的生物体， 从细菌到人类。 该项目的长期目标是 了解λ噬菌体如何进行位点特异性 重组 生物化学和遗传学方法将用于 表征蛋白质-蛋白质和蛋白质-DNA相互作用， 在重组复合物（整合体）的组装过程中， 链交换的过程。 噬菌体编码的整合酶（Int）蛋白 参与整合体形成并催化链交换。 将分离具有改变的DNA结合特异性的突变体， 确定Int中的哪些氨基酸残基负责DNA 识别. 其他Int突变体将在 确定单个突变体缺陷的生物化学测定 重组途径中的蛋白质。 宿主编码的集成宿主 因子（IHF）参与整合体的形成，诱导弯曲， DNA. 体内和体外诱变方法的组合将 用于分离和表征改变的DNA结合特异性 IHF的突变体。 这样的突变体将鉴定出 IHF与DNA相互作用，并将有助于解释结构 从物理研究中得出的。 噬菌体编码的切除酶（Xis）和 宿主编码的倒位刺激因子（FIS）启动子切除 重组 Xis与Int和FIS协同作用。 Xis 将分离出在协同相互作用中有缺陷的突变体 与Int和FIS和突变蛋白将用于确定 合作互动的机制。