Gyrase Binding Sites in Bacteriophage Mu Transposition and Chromosome Structure

Pato噬菌体Mu/由于其既是病毒又是转座子的性质，在转座子中是不寻常的，其体积大，转座子频率高。尽管受到细菌体复杂结构的限制，但在细菌类核内发生的一系列复制转位事件中，在裂解周期中扩增Mu DNA。由于担心在类核内连接前噬菌体DNA末端的潜在问题，这是转位过程的早期步骤，导致研究发现Mu基因组中心有一个强旋酶结合位点，这是有效复制所必需的。提出了一种假设，即螺旋酶位点参与组织超卷曲前噬菌体DNA的拓扑结构，以协助Mu末端的突触。该模型的几个预测在上一个拨款期间进行了测试，迄今为止的结果为该假设提供了强有力的支持。本研究继续了Mu的研究，并将其扩展到包括强回转酶结合位点在染色体结构中的可能作用的研究。我们将测试Mu强回转酶位点有助于将与回转酶位点等距离的相互作用DNA序列聚集在一起的预测，并检查这种活性所需的回转酶和结合位点的特征。一个强大的选择程序的分离和表征潜在的强旋酶结合位点从大肠杆菌染色体提出。染色体DNA片段将被克隆到原噬菌体的中心，因为它的旋切酶位点被删除，因此不能形成空斑；任何恢复斑块形成能力的片段都是携带强旋切酶位点的候选片段。本研究中对细菌和病毒复制的研究有可能为干扰其他生物体中的病毒生长提供有用的信息。*** ??

9420804 Pato Bacteriophage Mu/ by nature 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 Mu DNA during the lytic cycle occurs by a series of replicative transposition events which take place within the bacterial nucleoid, despite the restraints imposed by the complex structure of that body. Concerns about potential problems with 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 in the center of the Mu genome which is required for efficient replication. An hypothesis was proposed that the gyrase site is involved in organizing the topology of the supercoiled prophage DNA to assist in synapsis of the Mu ends. Several predictions of the model were tested during the previous grant period and the results to date offer strong support for the hypothesis. The present research continues the studies with Mu and extends them to include studies on the possible role of strong gyrase binding sites in chromosomal structure. The prediction that the Mu strong gyrase site can assist in bringing together interacting DNA sequences that are equidistant from the gyrase site will be tested/ and features of gyrase and of the binding site that are required for this activity will be examined. A powerful selection procedure for the isolation and characterization of potential strong gyrase binding sites from the E. coli chromosome is presented. Fragments of chromosomal DNA will be cloned into the center of a prophage deleted for its gyrase site, and hence unable to form a plaque; any fragment that restores plaque forming ability will be a candidate for one carrying a strong gyrase site. %%% Studies of bacterial and viral replication in this research have the potential to provide information useful in interfering with virus growth in other organisms. *** ??