Bacteriophage Mu transposition and role of gyrase binding sites

该项目将继续研究噬菌体Mu强旋转酶位点(SGS)，它是突触原噬菌体末端允许复制转位Mu DNA所必需的。正在研究的模型假设，位于中心的SGS通过组织原噬菌体DNA的拓扑结构形成一个超级卷曲的环，SGS在环的顶端，末端在底部，从而促进突触。SGS的生化研究表明，与旋转酶的高效结合与高效和进行性的超级卷曲相结合。基因解剖表明，SGS的右臂是它的区别元素。本项目关注两个重要问题：1)SGS如何促进Mu噬菌体末端的突触；2)为什么需要一种新的机制来促进Mu原噬菌体末端的突触。第一个问题将通过对旋转酶和SGS相互作用的联合遗传和生化分析来解决，以确定SGS的特征，特别是负责其独特特性的“臂”的作用。第二个问题将通过研究阻碍Mu原噬菌体末端生产性突触的因素来解决，重点是潜在的类核结构域屏障在将Mu末端分离为拓扑隔离结构域时的影响。这些研究的更广泛的影响，除了加深我们对Mu转座的理解外，还将为DNA旋转酶的生物化学提供见解，DNA旋转酶是细胞生物学中的一种关键酶，也是抗生素干预的主要目标，以及细菌核仁的结构。与当地一所高中和当地本科院校雷吉斯学院(Regis College)建立了外联计划，将在那里授课，感兴趣的学生将被带到实验室接受实践研究经验，同时为项目目标做出贡献。

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.