MECHANISTIC ANALYSIS OF DNA TRANSPOSITION BY PHAGE MU

转座重组是一种强大的遗传发生器， 多样性 一组完全不同的元素，与许多不同的 生物体，促进自己的运动从一个DNA位点到另一个， 换位 本项目的长期目标是了解 噬菌体Mu转座的分子机制 的非凡 Mu换位的效率使其成为机械的理想系统， 问题研究 转座的核心DNA切割和连接反应 是已知的。 Mu，细菌元件Tn 10和逆转录病毒使用 基本上相同的DNA切割和连接步骤，并且各自编码一个 促进这些反应的蛋白质。 最近，人们已经清楚地看到 Mu转座酶的活性形式是蛋白质的四聚体 绑定到元素的成对末端。 这一见解提供了一个 有机会分析多个DNA的切割和连接 反应在四聚体蛋白质复合物内协调。 通过 使用部分缺陷的转座酶和位点特异性DNA-蛋白 交联，这种活性复合物的功能组织将是 试探 这些实验，沿着额外的交联，化学交联， 修饰和诱变研究，也将定位和解剖 Mu转座酶的活性位点。 遗传和生物化学实验 为了解决Mu转座蛋白的作用， 因素，在招募细胞复制机制， 还提出了转座中间体。 反应的相对简单，蛋白质-DNA的稳定性 复合物，以及复合过程中对多个DNA位点的要求 组装，也使穆换位一个理想的系统，研究 蛋白质-DNA复合物组装的潜在机制。 理解 Mu转座中的蛋白质-DNA组装过程很可能 提供可推广到转录领域的见解， 复制控制 转座的生物化学描述应该 也有助于转座子介导的诱变的发展， 生物，这是不可行的。 此外，影响 转座重组对人类健康的影响是巨大的。 快速 抗生素耐药性水平传播主要是由于 转座因子移动，在广泛的宿主范围质粒和作为 接合转座子，在整个细菌种群。 此外 逆转录病毒，包括艾滋病毒，整合到宿主染色体上， 机制与Mu转位相同。 了解分子 这一重要过程的机制应有助于未来的设计或 发现可以防止这些不良后果的试剂， 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.