ANALYSES OF SITE SPECIFIC RECOMBINATION

位点特异性重组分析

• 批准号: 2634635
• 负责人: JEFFREY F GARDNER
• 金额: $ 27.32万
• 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 2000-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2634635
• 关键词: DNA binding protein; DNA topoisomerases; bacteriophage lambda; enzyme activity; gel mobility shift assay; gene complementation; gene mutation; genetic recombination; integrase; intermolecular interaction; microorganism genetics; molecular cloning; nucleic acid sequence; site directed mutagenesis

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 X carries-out site-specific recombination. The findings will have a significant impact on other site-specific recombination systems because many of their integrases are related to the lambda Int protein. Biochemical and genetic approaches will be used to characterize the protein-protein and protein-DNA interactions that occur both during the assembly of recombination complexes (intasomes) and the process of strand-cleavage and exchange. The phage-encoded integrase (Int) protein is a central actor in intasome formation and strand exchange. Int mutants will be isolated and characterized in assays that will determine the defects of the individual proteins in the recombination pathway. The host-encoded integration host factor (IHF) also participates in intasome formation by inducing bends in the DNA. We will isolate mutants that have amino acid substitutions of residues that, as suggested by our previous genetic studies, interact with DNA. Such mutants will also provide information that will be useful in interpreting data derived from physical studies on the protein. The phage-encoded excisionase (Xis) and the host- encoded factor for inversion stimulation (FIS) promote excisive recombination. Xis interacts cooperatively with Int and FIS. We will characterize the mechanism(s) of cooperative protein-protein interactions by isolating and characterizing Xis mutants that are defective in interacting with Int or FIS.

位点特异性重组系统对于控制非常重要 多种生物体的发育和基因表达 从细菌到人类。该项目的长期目标是 了解噬菌体 X 如何进行位点特异性重组。这 调查结果将对其他特定地点产生重大影响 重组系统，因为它们的许多整合酶都与 lambda Int 蛋白。生物化学和遗传学方法将用于 描述发生的蛋白质-蛋白质和蛋白质-DNA 相互作用 在重组复合物（整合体）的组装过程中和 链断裂和交换的过程。噬菌体编码的整合酶 (Int) 蛋白质是嵌体形成和链交换的核心参与者。 INT 突变体将被分离并在测定中进行表征，以确定 重组途径中单个蛋白质的缺陷。这 宿主编码的整合宿主因子（IHF）也参与整合体 通过诱导 DNA 弯曲而形成。我们将分离出具有 残基的氨基酸取代，正如我们之前所建议的 遗传学研究，与DNA相互作用。此类突变体还将提供 有助于解释物理数据的信息 对蛋白质的研究。噬菌体编码的切除酶 (Xis) 和宿主 反转刺激编码因子 (FIS) 促进切除 重组。 Xis 与 Int 和 FIS 合作互动。我们将 表征蛋白质-蛋白质相互作用的协同机制 通过分离和表征存在缺陷的 Xis 突变体 与 Int 或 FIS 交互。