MECHANISTIC ANALYSIS OF DNA TRANSPOSITION BY PHAGE MU

噬菌体Mu以非常高的频率转座， 转座的基础研究已经使用Mu完成。 现在 很明显，这种重组机制的关键方面是由穆 和许多其他的转座因子，它们侵入原核生物， 真核生物 该项目的长期目标是 了解Mu转座的分子机制。 它最近 很明显，Mu转座酶是一个结构相关的 包括许多转座酶和逆转录病毒的蛋白质家族 整合酶 这些蛋白质催化重组为多聚体 与DNA结合的复合物。 理解组织结构 转座酶-DNA复合物的最先进的Mu转座酶。 Mu转座酶的活性形式是同时结合的同源四聚体， 与参与重组的三个DNA片段相关联。 的 该提案的具体目标是提供一个全面的 描述DNA分子如何排列在这个四聚体中， 蛋白质复合物以及复合物是如何组装的。 我们会用蛋白质- DNA和蛋白质-蛋白质交联以绘制Mu区域 在重组位点与DNA相互作用的转座酶， 提供将四聚体保持在一起的蛋白质-蛋白质表面。 活性转座酶四聚体的组装完全取决于 蛋白质与特定DNA位点的结合; 这种DNA依赖性将使用多个探针进行研究， 蛋白质构象的变化。 遗传和生物化学实验 为了解决如何选择合适的DNA靶位点， 还提出了重组。 这种机制的分析 作为蛋白质-DNA复合物的组装和活化的基础 参与换位可能会提供深入了解 控制转录和复制的机制。 转基因对人类健康的影响是巨大的。 快速 抗生素耐药基因的传播主要是由于 转座因子在细菌群体中移动。 此外，逆转录病毒，包括艾滋病毒， 染色体通过几乎与转座相同的机制。 一个相关 重组反应也负责组装 免疫球蛋白和T细胞受体基因在发育过程中的作用 脊椎动物的免疫系统 了解这一现象的分子机制 重要的一类基因重组应该有助于未来的设计 或发现可以防止不良后果的试剂， 换位

Phage Mu transposes at an extraordinarily high frequency and many fundamental studies of transposition have been done using Mu. It is now clear that key aspects of this recombination mechanism are shared by Mu and many other transposable elements that invade prokaryotic and eukaryotic organisms. The long term goal of this project is to understand the molecular mechanism of Mu transposition. It has recently become clear that Mu transposase is a member of a structurally related family of proteins that includes many transposases and the retroviral integrases. These proteins catalyze recombination as multimeric complexes bound to DNA. Understanding of the structural organization of a transposase-DNA complex in most advanced for the Mu transposase. The active form of Mu transposase is a homotetramer bound simultaneously to the three segments of DNA that participate in recombination. The specific goals of this proposal are aimed at providing a thorough description of how the DNA molecules are arranged within this tetrameric protein complex and how the complex is assembled. We will use protein- DNA and protein-protein cross-linking to map the regions of Mu transposase that interact with the DNA at the recombination sites and provide the protein-protein surfaces that hold the tetramer together. Assembly of the active transposase tetramer absolutely depends on binding of the protein to specific DNA sites; the structural basis of this DNA-dependence will be investigated using multiple probes for changes in protein conformation. Genetic and biochemical experiments to address how an appropriate DNA target site is selected during recombination are also proposed. This analysis of the mechanisms underlying the assembly and activation of the protein-DNA complexes involved in transposition is likely to provide insights into the mechanisms that control transcription and replication as well. The impact of transposition on human health is immense. The rapid spread of antibiotic resistance genes is largely a result of transposable elements moving throughout bacterial populations. Furthermore, retroviruses, including HIV, integrate into the host chromosome via a mechanism nearly identical to transposition. A related recombination reaction is also responsible for assembly of the immunoglobulin and T-cell receptor genes during development of the vertebrate immune system. Understanding the molecular mechanism of this important class of genetic recombination should assist the future design or discovery of agents that may prevent the undesirable consequences of transposition.