Bacteriophage Mu transposition and role of gyrase binding sites

University Of Colorado At Denver, Aurora CO

Investigators

Abstract

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.