Bacteriophage Mu transposition and role of gyrase binding sites

该项目将继续研究噬菌体Mu强促旋酶位点（SGS），该位点是突触前噬菌体末端允许Mu DNA复制转座所需的。研究的模型假定，位于中央的SGS通过组织原噬菌体DNA的拓扑结构形成超螺旋环，SGS在环的顶端，末端在基部，从而促进突触。 与SGS的生化研究表明，高效结合到旋转酶，再加上高效和进行性超螺旋。 遗传解剖暗示SGS的右“臂”是其区别元素。 本项目集中在两个重要的问题：1）SGS如何在促进Mu前噬菌体末端的突触中发挥作用，以及2）为什么需要一种新的机制来促进Mu前噬菌体末端的突触。第一个问题将解决的促旋酶和SGS的相互作用，以确定SGS的功能，特别是“武器”的作用，这是负责其独特的性能相结合的遗传和生物化学分析。第二个问题将通过研究可以阻碍Mu原噬菌体末端的生产性突触的因素来解决，重点关注潜在的类核结构域障碍在将Mu末端分离成拓扑隔离结构域中的影响。 这些研究的更广泛的影响，除了促进我们对Mu转座的理解外，还将为DNA促旋酶的生物化学提供见解，DNA促旋酶是细胞生物学中的一种关键酶，也是抗生素干预的主要靶点，以及细菌类核的结构。 与当地一所高中和当地一所本科院校里吉斯学院建立了外联方案，在那里将举行讲座，感兴趣的学生将被带到实验室，获得实践研究经验，同时为项目的目标做出贡献。

The project will continue studies of the bacteriophage Mu strong gyrase site (SGS) that is required for synapsing prophage termini to allow replicative transposition of Mu DNA. The model under study postulates that the centrally located SGS promotes synapsis by organizing the topology of prophage DNA to form a supercoiled loop with the SGS at the apex of the loop and the termini at the base. Biochemical studies with the SGS showed highly efficient binding to gyrase coupled with highly efficient and processive supercoiling. Genetic dissection implicated the right "arm" of the SGS as its distinguishing element. The present project focuses on two important questions: 1) How does the SGS function in promoting synapsis of the Mu prophage ends, and 2) Why is a novel mechanism required for promoting the synapsis of Mu prophage ends. The first question will be addressed by a combined genetic and biochemical analysis of the interaction of gyrase and the SGS to determine the features of the SGS, particularly the roles of the "arms", that are responsible for its unique properties. The second question will be addressed by examining factors that can impede productive synapsis of Mu prophage termini, focusing on the effects of potential nucleoid domain barriers in separating Mu ends into topologically isolated domains. Broader impact of these studies, in addition to furthering our understanding of Mu transposition, will be to provide insights into the biochemistry of DNA gyrase, a critical enzyme in cell biology and a major target for antibiotic intervention, and into the structure of the bacterial nucleoid. Outreach programs have been established with a local high school and with Regis College, a local undergraduate institution, where lectures will be given, and interested students will be brought into the laboratory to receive hands-on research experience while contributing to the goals of the project.