MECHANISTIC ANALYSIS OF DNA TRANSPOSITION BY PHAGE MU

易位重组是一种强大的基因生成器 多样性。一组完全不同的元素，与许多不同的 生物体通过以下方式促进自身从一个DNA位点到另一个DNA位点的移动 换位。该项目的长期目标是了解 噬菌体Mu转座的分子机制。不平凡的 Mu换位的效率使其成为一种理想的机械系统 学习。转座中心的DNA切割和连接反应 都是已知的。Mu，细菌元件Tn10，和逆转录病毒使用 基本上相同的DNA切割和连接步骤，每个步骤都编码一个 促进这些反应的蛋白质。最近，情况变得明朗起来 Mu转座酶的活性形式是蛋白质的四聚体 绑定到元素的成对两端。这种洞察力提供了一种 分析多个DNA如何切割和连接的机会 在四聚体蛋白质复合体中，反应是协调的。通过 利用部分有缺陷的转座酶和位点特异性DNA蛋白 交联剂，这种活性复合体的功能组织将是 探查过了。这些实验，加上额外的交联剂，化学 修饰和突变研究也将定位和剖析 Mu转座酶的活性部位。遗传和生化实验 为了解决Mu转座蛋白的作用，以及特定的宿主 在将细胞复制机制招募到 并提出了转位中间体的概念。 反应相对简单，蛋白质-DNA的稳定性 复合体，以及复合体过程中对多个DNA位点的要求 组装，也使穆易位成为研究的理想系统。 蛋白质-DNA复合体组装的潜在机制。理解 Mu转座中蛋白质-DNA组装过程的 提供可概括到转录和 复制控制。转位的生化描述应该是 也促进了转座子介导的突变在 在这方面尚不可行的生物。此外， 转位重组对人类健康的影响是巨大的。The Rapid 抗生素耐药性的水平传播在很大程度上是 转座元件在广泛寄主范围的质粒和AS上移动 接合转座子，遍及细菌种群。更有甚者 逆转录病毒，包括艾滋病毒，通过一种 机制与Mu转位相同。理解分子 这一重要过程的机械应有助于今后的设计或 发现可以防止这些不良后果的药物 换位。

Transpositional recombination is a powerful generator of genetic diversity. A disparate set of elements, isolated from many different organisms, promote their own movement from one DNA site to another by transposition. The long term goal of this project is to understand the molecular mechanism of transposition by phage Mu. The extraordinary efficiency of Mu transposition makes it an ideal system for mechanistic studies. The DNA cleavage and joining reactions central to transposition are known. Mu, the bacterial element Tn10, and retroviruses use essentially the same DNA cleavage and joining steps, and each encodes a protein that promotes these reactions. Recently, it has become clear that the active form of the Mu transposase is a tetramer of the protein bound to the paired ends of the element. This insight provides an opportunity to analyze how the multiple DNA cleavage and joining reactions are coordinated within the tetrameric protein complex. By using partially defective transposases and site specific DNA-protein crosslinking, the functional organization of this active complex will be probed. These experiments, along with additional crosslinking, chemical modification, and mutagenesis studies, will also locate and dissect the active sites of the Mu transposase. Genetic and biochemical experiments to address the role of the Mu transposition proteins, and specific host factors, in recruiting the cellular replication machinery to the transposition intermediate are also proposed. The relative simplicity of the reaction, the stability of the protein-DNA complexes, and the requirement for multiple DNA sites during complex assembly, also make Mu transposition an ideal system to study the mechanisms underlying assembly of protein-DNA complexes. Understanding of the protein-DNA assembly process in Mu transposition is likely to provide insights generalizable to the fields of transcription and replication control. A biochemical description of transposition should also facilitate development of transposon-mediated mutagenesis in organisms where this is not yet feasible. In addition, the impact of transpositional recombination on human health is immense. The rapid horizontal spread of antibiotic resistance is largely a result of transposable elements moving, on broad host range plasmids and as conjugative transposons, throughout bacterial populations. Furthermore retroviruses, including HIV, integrate into the host chromosome by a mechanism identical to Mu transposition. Understanding the molecular mechanism of this important process should assist the future design or discovery of agents that may prevent these undesirable consequences of transposition.