ANALYSES OF SITE SPECIFIC RECOMBINATION

位点特异性重组分析

• 批准号: 2857090
• 负责人: JEFFREY F GARDNER
• 金额: $ 28.13万
• 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/2857090
• 关键词: DNA binding protein; DNA topoisomerases; bacteriophage lambda; enzyme activity; gel mobility shift assay; gene complementation; gene mutation; genetic recombination; integrase; microorganism genetics; molecular cloning; nucleic acid sequence; protein protein interaction; 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 了解噬菌体X如何进行位点特异性重组。的 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与重组系统相关。 λ Int蛋白。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， 链断裂和交换的过程。噬菌体编码整合酶（Int） 蛋白质是整合体形成和链交换的中心作用物。Int 突变体将被分离并在测定中表征， 重组途径中单个蛋白质的缺陷。的 宿主编码的整合宿主因子（IHF）也参与整合体的形成。 通过诱导DNA弯曲来形成。我们会隔离那些 如我们先前的研究所建议的， 基因研究，与DNA相互作用。这些突变体还将提供 这些信息将有助于解释来自物理 研究蛋白质。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协同作用。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Xis突变体， 与Int或FIS交互。