Analyses of Site-Specific Recombination

描述（由申请人提供）：位点特异性重组系统是 在控制这些不同的过程如质粒维持中是重要的， DNA扩增、染色体分离、接合转座子的运动 和基因在整合子中的整合。这项研究的长期目标是 了解噬菌体Lambda Int如何进行位点特异性重组。 研究结果将对其他特定地点产生重大影响 这些重组系统是不同的Lambda Int家族的成员。 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， 重组复合物（整合体）的组装和链的过程 切割和连接。Int突变体将在测定中表征， 旨在确定重组中单个蛋白质的缺陷， 通路宿主编码的整合宿主因子（IHF）也参与其中 通过诱导DNA弯曲形成整合体。我们将结晶突变体 这些蛋白质具有扩展的识别特异性， 蛋白质内氨基酸的相互作用如何介导DNA的识别。 位点特异性切除反应对于来自 一个基因组到另一个。我们将分析切除酶（Xis） 蛋白质与INt和DNA除了噬菌体λ（Xis）蛋白之外， 关于Xis蛋白在切除反应期间如何起作用知之甚少。 为了扩大我们对切除反应复合物的理解， 反应中，我们计划分析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的项目， 转座子CTnDOT。我们的初步工作表明，虽然CTnDOT有一个 与Lambda Int相关的整合酶，它似乎有一些 机械差异。我们计划开发一个体外系统来研究 CTnDOT催化的链交换和切除反应的机理 Int蛋白。

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.