ANALYSES OF SITE SPECIFIC RECOMBINATION

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X是如何进行定点重组的。这个 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 Lambda Int蛋白质。将使用生化和遗传方法来 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)的组装过程中和在 链断裂和交换的过程。噬菌体编码整合酶(Int) 蛋白质是内含体形成和链交换的中心角色。整型 突变株将被分离并进行鉴定，以确定 重组途径中单个蛋白质的缺陷。这个 宿主编码的整合宿主因子(IHF)也参与了内含体 通过诱导DNA中的弯曲而形成。我们将分离出具有 氨基酸取代的残基，如我们以前的建议 基因研究，与DNA相互作用。这样的突变体还将提供 在解释从物理数据派生的数据时有用的信息 对蛋白质的研究。噬菌体编码的摘除酶(XIS)和宿主- 反转刺激编码因子(FIS)促进兴奋 重组。XIS与Int和FIS协同交互。我们会 描述蛋白质-蛋白质相互作用的协同机制(S) 通过分离和鉴定存在缺陷的XIS突变体 与Int或FIS交互。

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.