MECHANISTIC ANALYSIS OF DNA TRANSPOSITION BY PHAGE MU

噬菌体Mu以极高的频率和许多 利用Mu进行了转位的基础研究。现在是时候了 明确这种重组机制的关键方面为Mu所共享 和许多其他入侵原核生物的转座子 真核生物。这个项目的长期目标是 了解Mu转位的分子机制。最近是这样的 很明显，Mu转座酶是结构上相关的 包括许多转座酶和逆转录病毒的蛋白质家族 整合酶。这些蛋白质以多聚体的形式催化重组 与DNA结合的复合体。对结构性组织的理解 转座酶-DNA复合体中最高级的为Mu转座酶。 Mu转座酶的活性形式是同时结合的同源四聚体 到参与重组的三个DNA片段。这个 这项提案的具体目标旨在提供全面的 描述DNA分子在这个四聚体中是如何排列的 蛋白质复合体以及复合体是如何组装的。我们将使用蛋白质- DNA和蛋白质-蛋白质交联法定位小鼠的区域 在重组位点与DNA相互作用的转座酶 提供将四聚体结合在一起的蛋白质-蛋白质表面。 活性转座酶四聚体的组装完全依赖于 蛋白质与特定DNA位点的结合；结构基础 这种对DNA的依赖将使用多个探针进行调查 蛋白质构象的变化。遗传和生化实验 要解决如何在选择合适的DNA目标位置 此外，还提出了复合的设想。这一机制的分析 蛋白质-DNA复合体的组装和激活 可能会为我们提供对 控制转录和复制的机制。 移位对人类健康的影响是巨大的。The Rapid 抗生素耐药性基因的传播在很大程度上是由 在细菌种群中移动的转座子。 此外，包括艾滋病毒在内的逆转录病毒会整合到宿主体内 染色体通过一种与转座几乎相同的机制。A相关 重组反应也负责组装 免疫球蛋白和T细胞受体基因在儿童发育中的作用 脊椎动物的免疫系统。了解这一现象的分子机制 一类重要的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制剂 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.