Bacteriophage Mu transposition and role of gyrase binding sites

该项目将继续研究噬菌体Mu强旋酶位点（SGS），这是连接前噬菌体末端以允许Mu DNA复制转位所必需的。本研究模型假设，位于中心的SGS通过组织前噬菌体DNA的拓扑结构形成一个超螺旋环，SGS位于环的顶端，末端位于环的底部，从而促进突触的形成。用SGS进行的生化研究表明，它与旋切酶具有高效的结合和高效的超卷曲。遗传解剖暗示了SGS的右臂是其区别元素。本项目主要关注两个重要问题：1)SGS在促进Mu前噬菌体末端突触中的作用如何；2)为什么需要一种促进Mu前噬菌体末端突触的新机制。第一个问题将通过对gyrase和SGS相互作用的综合遗传和生化分析来解决，以确定SGS的特征，特别是“臂”的作用，这是其独特特性的原因。第二个问题将通过研究可能阻碍Mu噬菌体末端产生突触的因素来解决，重点关注潜在的类核结构域屏障在将Mu末端分离到拓扑隔离结构域中的作用。这些研究的广泛影响，除了进一步加深我们对Mu转位的理解外，还将为DNA回转酶（细胞生物学中的关键酶和抗生素干预的主要靶点）的生物化学以及细菌类核结构提供见解。与当地的一所高中和瑞吉斯学院（当地的一所本科院校）建立了外展计划，在那里将进行讲座，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.