Bacteriophage Mu Transposition and the Role of Gyrase Binding Sites

细菌病毒Mu由于其既是病毒又是转座子，在转座子中以其大尺寸和高转座频率而不寻常。在病毒生长周期中，37.2 kb Mu DNA的扩增通过一系列发生在细菌类核（DNA和相关蛋白质）内的复制性转座事件发生，尽管该体的复杂结构施加了限制。对参与类核内原噬菌体DNA末端突触的长程DNA相互作用的兴趣，转座过程的早期步骤，导致了在Mu基因组中心发现有效复制所需的强促旋酶结合位点（SGS）的研究。早期的模型假设SGS促进Mu原噬菌体末端的突触，并且它通过组织超螺旋原噬菌体DNA的拓扑结构以形成在环的顶点处具有SGS并且在基部处具有末端的plectonemically interwound环来实现。 该模型的预测实验测试提供了强有力的支持，建议的SGS的作用。虽然在第二个转座病毒中发现了类似的位点，但广泛的搜索没有发现可以在Mu复制中取代SGS的染色体或质粒位点。为了确定该位点的独特特征，对SGS进行了遗传分析。鉴定了促旋酶位点的“核心”下游区域，其负责赋予SGS促进Mu复制的能力。 该项目重点关注该模型提出的两个重要问题：1）为什么需要一种新的机制来促进Mu前噬菌体末端的突触; 2）SGS如何在促进Mu前噬菌体末端的突触中发挥作用？ 第一个问题将通过探索在没有SGS的情况下造成Mu复制长时间延迟的因素来解决。最近开发的一种灵敏的检测方法，用于检测诱导溶原后的第一轮转座的早期阶段，将被用来检查的因素，包括由细菌类核的结构域结构所施加的限制，转录的拓扑后果，和穆和主机编码的蛋白质的作用。 第二个问题将通过SGS的结构和功能分析来解决，重点是下游区域，该区域赋予SGS促进复制的能力。分析将包括研究促旋酶与SGS和其他促旋酶位点的结合，以及研究SGS右臂的遗传改变对原噬菌体末端突触的影响。 这些研究将进一步了解Mu复制转座的机制，并提供对细菌类核结构（DNA必须压缩数千倍才能适应细菌细胞，但仍然必须能够复制和转录）和DNA促旋酶生物化学的见解。 利用这种遗传上易于处理的系统，应该可以深入了解植物和动物的转座子和病毒，它们可以进出宿主的染色体。

Bacterial virus Mu, by virtue of its being both a virus and a transposon, is unusual among transposons in its large size and its high frequency of transposition. Amplification of the 37.2 kb Mu DNA during the virus growth cycle occurs by a series of replicative transposition events which take place within the bacterial nucleoid (DNA and associated proteins), despite the constraints imposed by the complex structure of that body. Interest in the long-range DNA interactions involved in synapsing the ends of prophage DNA within the nucleoid, an early step in the transposition process, led to studies which uncovered a strong gyrase binding site (SGS) in the center of the Mu genome that is required for efficient replication. An earlier model postulated that the SGS promotes synapsis of the Mu prophage termini, and that it does so by organizing the topology of the supercoiled prophage DNA to form a plectonemically interwound loop with the SGS at the apex of the loop and the termini at the base. Experimental tests of predictions of the model have offered strong support for the proposed role of the SGS. While a similar site was found in a second transposing virus, an extensive search found no chromosomal or plasmid sites which could replace the SGS in Mu replication. To determine the unique features of the site, a genetic analysis of the SGS was performed. A region downstream of the "core" of the gyrase site was identified that is responsible for imparting to the SGS the ability to promote Mu replication. This project focuses on two important questions raised by the model: 1) Why is a novel mechanism required for promoting the synapsis of Mu prophage ends; and 2) How does the SGS function in promoting synapsis of the Mu prophage ends? The first question will be addressed by exploring the factors that are responsible for the long delay in Mu replication in the absence of the SGS. A recently developed sensitive assay for detecting the early stages of the first round of transposition after induction of a lysogen will be used to examine factors including the constraints imposed by the domain structure of the bacterial nucleoid, the topological consequences of transcription, and the role of Mu and host encoded proteins. The second question will be addressed by a combined structural and functional analysis of the SGS, with emphasis on the downstream region which imparts to the SGS the ability to promote replication. The analysis will include studies of the binding of gyrase to the SGS and to other gyrase sites and studies on the effects of genetic alterations of the SGS right arm on synapsis of prophage ends. These studies will further understanding of the mechanism of Mu replicative transposition and provide insights into the structure of the bacterial nucleoid (the DNA has to be compacted several thousand fold to fit into the bacterial cell, yet still has to be able to replicate and be transcribed) and the biochemistry of DNA gyrase. Work with this genetically tractable system should provide insight into transposons and viruses of plants and animals that move in and out of their hosts' chromosomes.