Analyses of Site-Specific Recombination

描述(申请人提供)：特定部位的重组系统有 在控制这样的不同过程中很重要，例如质粒维持， DNA扩增、染色体分离、接合转座子运动 以及整合子中基因的整合。这项研究的一个长期目标是 了解噬菌体Lambda Int如何进行定点重组。 这些发现将对其他特定地点产生重大影响 重组系统是不同的Lambda Int家族的成员。 将使用生化和遗传方法来表征 蛋白质-蛋白质和蛋白质-DNA的相互作用 重组复合体(内含体)的组装和链的加工 卵裂和结扎。INT突变体将在符合以下条件的检测中进行表征 用来确定重组过程中个别蛋白质的缺陷 路径。主机编码的整合主机因子(IHF)也参与 通过在DNA中诱导弯曲来形成肠小体。我们将使变种人具体化 具有扩展的识别特异性以便理解的蛋白质 蛋白质中氨基酸的相互作用如何调节DNA的识别。 特定部位的切除反应对于元素的传播非常重要 从一个基因组到另一个基因组。我们将分析切除酶(XIS)的相互作用 含有INT和DNA的蛋白质。除噬菌体Lambda(Xis)蛋白外， 关于XIS蛋白在切除反应中如何发挥作用，人们知之甚少。 为了扩大我们对切除反应络合物的理解和 反应，我们计划分析PAGE的XIS功能的分子机制 P22 Xis蛋白。最后，我们将启动一个关于共轭词的项目 转座子CTnDOT。我们的初步工作表明，尽管CTnDOT有一个 与Lambda Int相关的整合酶，它似乎有一些 机械上的差异。我们计划开发一个体外系统来研究 链交换机理及CTnDOT催化的切割反应 INT蛋白质。

DESCRIPTION (provided by applicant): Site-specific recombination systems are important in controlling such diverse processes such as plasmid maintenance, DNA amplification, chromosome segregation, movement of conjugative transposons and integration of genes in integrons. A long-range goal of this research is to understand how bacteriophage Lambda Int performs site-specific recombination. The findings will have a significant impact on other site-specific recombination systems that are members of the diverse Lambda Int family. 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 processes of strand cleavage and ligation. Int mutants will be characterized in assays that are designed to determine the defects of individual proteins in the recombination pathway. The host-encoded integration host factor (IHF) also participates in formation of intasomes by inducing bends in the DNA. We will crystallize mutant proteins that have expanded recognition specificities in order to understand how interactions of amino acids within the protein mediate recognition of DNA. Site-specific excision reactions are important for the spread of elements from one genome to another. We will analyze interactions of the excisionase (Xis) protein with INt and DNA. With the exception of phage Lambda (Xis) protein, little is known about how Xis proteins function during the excision reaction. In order to expand our understanding of excision reaction complexes and reactions, we plan to analyze the molecular mechanism of Xis function of page P22 Xis protein. Finally, we will initiate a project on the conjugative transposon CTnDOT. Our preliminary work indicates that although CTnDOT has an integrase that is related to the Lambda Int, it appears to have some mechanistic differences. We plan to develop an in vitro system to study the mechanism of strand exchange and the excision reaction catalyzed by the CTnDOT Int protein.